1. Developmental Biology
  2. Chromosomes and Gene Expression
Download icon

A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines

  1. Frederic Strobl  Is a corresponding author
  2. Anita Anderl
  3. Ernst HK Stelzer  Is a corresponding author
  1. Goethe Universität, Germany
Tools and Resources
  • Cited 1
  • Views 1,985
  • Annotations
Cite this article as: eLife 2018;7:e31677 doi: 10.7554/eLife.31677

Abstract

Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.

https://doi.org/10.7554/eLife.31677.001

eLife digest

Researchers frequently use model organisms, such as mice, zebrafish and various insect species, to understand biological processes – with the underlying idea that discoveries made can be applied to other species too. A common technique is genetic manipulation, in which a foreign gene is inserted into the chromosome of an organism. These introduced genes are called transgenes and the organisms carrying them are referred to as transgenic. Transgenic organisms are powerful tools to analyze biological processes or mimic human diseases.

Many model organisms carry two homologous chromosomes – one inherited from each parent. Pairs of chromosomes carry genes in the same order, but do not necessarily have identical versions of those genes. Newly created transgenic organisms, however, carry the transgene on only one of the chromosomes. This can be a problem for researchers, as many experiments require individuals that carry the transgene on both. Unfortunately, only costly and error-prone methods can distinguish between these individuals.

To overcome these drawbacks, Strobl et al. developed a concept called AGameOfClones and applied it to the red flour beetle Tribolium castaneum. In their approach, the transgene also expresses two marker-proteins with different fluorescent colors. After several generations of breeding, two versions of the transgene emerge – each retaining only one of the markers. This means that in the following generation, descendants that express both markers must be the offspring that carry the transgene on both of the chromosomes.

The AGameOfClones concept has several major advantages: individuals with different markers can be easily identified, the procedure is cost-efficient and reliable, and it can be applied to nearly all model organisms. This will benefit breeding schemes and animal welfare since irrelevant individuals can be excluded as soon as the markers become detectable.

https://doi.org/10.7554/eLife.31677.002

Introduction

Life sciences, especially cell and developmental biology, rely on model organisms. The most frequently used vertebrates are mouse and zebrafish. Amongst insects, the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum are the two prevailing species. An important standard technique is transgenesis, that is, the insertion of recombinant DNA into the genome of the model organism (Gama Sosa et al., 2010). Since model organisms are typically diploid, the genotype has to be considered, which leads to a certain experimental complexity. Usual mating schemes result in (i) non-transgenic wild-type progeny, (ii) hemizygous transgenic progeny, that is, only the maternal or only the paternal chromosome carries the transgene, and (iii) homozygous transgenic progeny, that is, both the maternal and paternal chromosomes carry the transgene. In rare cases, the phenotype reveals the genotype, but usually, either two of the three or even all three outcomes cannot be distinguished. Transformation markers can be used to separate wild-type from transgenic, but not hemi- from homozygous individuals. Thus, additional experiments are necessary to determine the genotype, for example genetic assays, which are invasive and require manpower as well as consumables.

In our AGameOfClones (AGOC) vector concept, all genotypes are directly identifiable by specifically designed distinct phenotypes, which permits the systematic creation of homozygous transgenic lines. Our approach relies on two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination results in hemizygous individuals that retain only one of the two markers and are thus phenotypically distinguishable from each other and the wild type. In the next generation, descendants that express both markers are identified as heterozygous for the transgene. Finally, a cross of two heterozygotes results in homozygous progeny that are selected by the lack of one marker.

Results

Proof-of-principle in the emerging insect model organism Tribolium castaneum

The proof-of-principle of the AGOC vector concept relied on the red flour beetle Tribolium castaneum, an emerging insect model organism (Klingler, 2004; Brown et al., 2009), in conjunction with the piggyBac transposon system (Lorenzen et al., 2003; Berghammer et al., 2009), which allows semi-random genomic insertion. We developed the transformation-ready pAGOC vector (Figure 1—figure supplement 1) that contains mOrange-based (Shaner et al., 2008) and mCherry-based (Shaner et al., 2004) eye-specific (Berghammer et al., 1999) transformation markers (mO and mC, respectively). Both fluorescent proteins are spectrally separable by appropriate excitation bands and emission filters (Shaner et al., 2005). Each marker is flanked upstream by a LoxP site (Hamilton and Abremski, 1984) and downstream by a LoxN (Livet et al., 2007) site, resulting in interweaved Lox site pairs (Figure 1). Due to variations in the spacer sequences, LoxP and LoxN sites are incompatible with each other.

Figure 1 with 4 supplements see all
The AGameOfClones vector concept within the piggyBac-based transformation-ready pAGOC vector for Tribolium.

Two fluorescence-based transformation markers, mO and mC, are embedded into a piggyBac-based transformation-ready vector, which is characterized by 3’ and 5’ terminal repeats (TR) necessary for genomic insertion. The markers are based on the artificial eye-specific 3×P3 promoter, the open-reading frame for the respective fluorescent protein, that is, mOrange or mCherry, and the SV40 poly(A). Each transformation marker is flanked upstream by a LoxP site (P) and downstream by a LoxN site (N), forming interweaved Lox site pairs. The markers can be detected in the eyes by using appropriate filter sets (FS). Cre-mediated recombination leads to the excision of one marker from the genome. Upon removal, the other marker remains within the genome, since the remaining LoxP and LoxN sites are incompatible. Individuals that underwent recombination give rise to progeny in which only one marker is detected in the eyes.

https://doi.org/10.7554/eLife.31677.003

We injected this vector together with a piggyBac transposase-expressing helper vector (that is, pATub’piggyBac) into pre-blastoderm embryos to achieve germline transformation. All survivors, that is, F1 potential mosaics, were mated with wild types and in six of these crosses, at least one F2 (mO-mC) founder female was found among the progeny. For each cross, one founder female was mated with a wild-type male and the progeny were scored to confirm that only a single insertion had occurred (Supplementary file 1). Transgenic descendants were collected to establish six proof-of-principle cultures, which carry the same transgene, but in different genomic locations. These F3 (mO-mC) pre-recombination hemizygous sublines were called AGOC #1 to #6. To roughly estimate homozygous viability, two F3 (mO-mC) pre-recombination hemizygous siblings were mated and the progeny were scored (Supplementary file 2). Additionally, the insertion locations of the transgenes were determined in four of the six AGOC sublines (Supplementary file 3). Up to this step, our scheme did not differ from most standard procedures to establish transgenic lines.

Systematic creation of homozygous transgenic lines

The mating procedure for the systematic creation of homozygous transgenic lines (Figure 2, an comprehensive scheme is provided in Figure 2—figure supplement 1) spanned four generations and involved a transgenic helper line, ICE{HSP68’NLS-Cre} #1. This line expresses a nuclear-localized Cre recombinase (Peitz et al., 2002) under control of the heat shock protein 68b promoter (Schinko et al., 2012) and carries a mCerulean-based (Markwardt et al., 2011) eye-specific transformation marker (mCe). The procedure was performed with all six AGOC sublines and phenotypically documented for #5 and #6 (Figure 3).

Figure 2 with 2 supplements see all
The AGameOfClones F3 to F7 mating procedure for the systematic creation of homozygous transgenic Tribolium lines.

A rounded rectangle illustrates the genotype for two independent autosomes, white bars represent the AGOC transgene location and black bars the Cre recombinase-expressing helper transgene location. A F2 (mO-mC) founder female × wild-type male cross gives rise to F3 (mO-mC) pre-recombination hemizygotes that carry mO and mC in cis configuration. A F3 (mO-mC) pre-recombination hemizygous female × mCe homozygous helper male cross results in F4 (mCe; mO-mC) double hemizygotes, in which one marker is removed through Cre-mediated recombination. Next, a F4 (mCe; mO-mC) double hemizygous female × wild-type male cross gives rise to F5 (mO) and (mC) post-recombination hemizygotes. A F5 (mO) post-recombination hemizygous female × F5 (mC) post-recombination hemizygous male sibling cross results in F6 (mO/mC) heterozygous progeny that carry mO and mC in trans configuration. Finally, a F6 (mO/mC) heterozygous female × a F6 (mO/mC) heterozygous male sibling cross gives rise to F7 (mO/mO) and (mC/mC) homozygous progeny. The percentage boxes indicate the theoretical ratio of the progeny that carry the respective genotype, the dashed line represents genotypically identical siblings. FS, filter set; rec, recombination; db, double.

https://doi.org/10.7554/eLife.31677.008
Figure 3 with 2 supplements see all
The AGameOfClones F3 to F7 mating procedure demonstrated for the AGOC #5 and #6 sublines.

From the F3 to the F7 generation, the genotype was phenotypically determined by monitoring mCe, mO and mC. For both sublines, F7 (mO/mO) and (mC/mC) homozygotes were obtained by following the mating procedure outlined in Figure 2. The wild-type male in the second row functions as the marker control. The percentage boxes indicate the experimental (and theoretical) ratio of the progeny that displayed the respective phenotype. FS, filter set; rec, recombination; db, double.

https://doi.org/10.7554/eLife.31677.011
  1. F3 (mO-mC) pre-recombination hemizygous females, which carried mO and mC on the maternal chromosome in cis configuration, were mated with (mCe/mCe) homozygous helper males (Figure 2 and Figure 3, first row). This resulted in F4 (mCe; mO-mC) double hemizygotes in which Cre-mediated recombination occurs (Table 1, F3 row). In this hybrid generation, adults displayed a patchy expression of mO and mC within their compound eyes (Figure 3—figure supplement 1).

  2. F4 (mCe; mO-mC) double hemizygous females were mated with wild-type males (Figure 2 and Figure 3, second row). Due to Cre-mediated recombination in the germline, this resulted in F5 (mO) and (mC) post-recombination hemizygotes that carried either only mO or only mC on the maternal chromosome (Table 1, F4 row).

  3. F5 (mO) post-recombination hemizygous females were mated with F5 (mC) post-recombination hemizygous male siblings (Figure 2 and Figure 3, third row), which resulted in F6 (mO/mC) heterozygotes that carried mO on the maternal and mC on the paternal chromosome in trans configuration (Table 1, F5 row). This was demonstrated by mating F6 (mO/mC) heterozygous females with wild-type males and scoring the progeny (Table 1, F6-S row).

  4. F6 (mO/mC) heterozygous females were mated with genotypically identical male siblings (Figure 2 and Figure 3, fourth row), which resulted in F7 (mO/mO) and (mC/mC) homozygotes that carried either only mO or only mC on both, the maternal and paternal chromosome (Figure 2 and Figure 3, fifth row as well as Table 1, F6 row). This was demonstrated by mating F7 (mO/mO) and (mC/mC) homozygous females with wild-type males and scoring the progeny (Table 1, F7-O and F7-C row, respectively).

Table 1
Mating procedure results for the six proof-of-principle AGOC sublines from the F3 to the F7 generation.

Bold entries mark progeny that were used in the subsequent cross. F6-S, F7-O and F7-C are control crosses. No significant differences between the arithmetic means and the theoretical Mendelian ratios were found. See Source Data 1 for raw scores ordered by transgenic sublines.

https://doi.org/10.7554/eLife.31677.014
GenCrossSublineProgeny
Total
F3Theoretical-50.0%-----50.0%-
AGOC #1-47.9% (46)-----52.1% (50)96
AGOC #2-52.4% (44)-----47.6% (40)84
AGOC #3-55.1% (49)-----44.9% (40)89
AGOC #4-44.1% (41)-----55.9% (52)93
AGOC #5-47.6% (39)-----52.4% (43)82
AGOC #6-48.2% (27)-----51.8% (29)56
Mean-49.2 ± 3.9%-----50.8 ± 3.9%83.3
F4Theoretical25.0%25.0%12.5%12.5%12.5%12.5%---
AGOC #125.6% (30)34.2% (40)4.3% (5)10.2% (12)6.0% (7)19.7% (23)--117
AGOC #227.3% (36)28.9% (38)15.1% (20)8.3% (11)9.8% (13)10.6% (14)--132
AGOC #325.8% (33)32.8% (42)13.3% (17)10.1% (13)5.5% (7)12.5% (16)--128
AGOC #4*14.0% (14)15.0% (15)20.0% (20)13.0% (13)22.0% (22)9.0% (9)3.0% (3)4.0% (4)100
AGOC #517.1% (20)40.2% (47)16.2% (19)8.5% (10)10.3% (12)7.7% (9)--117
AGOC #633.6% (39)33.6% (39)8.6% (10)6.9% (8)6.1% (7)11.2% (13)--116
Mean23.9 ± 7.2%30.8 ± 8.5%12.9 ± 5.6%9.5 ± 2.1%9.9 ± 6.3%11.8 ± 4.2%0.5%0.7%118.3
F5Theoretical25.0%-25.0%25.0%--25.0%--
AGOC #127.2% (31)-26.3% (30)23.7% (27)--22.8% (26)-114
AGOC #228.9% (26)-33.3% (30)17.8% (16)--20.0% (18)-90
AGOC #324.8% (30)-27.3% (33)24.8% (30)--23.1% (28)-121
AGOC #419.3% (21)-22.9% (25)37.6% (41)--20.2% (22)-109
AGOC #528.2% (31)-29.1% (32)12.7% (14)--30.0% (33)-110
AGOC #626.2% (22)-29.7% (25)16.7% (14)--27.4% (23)-84
Mean25.8 ± 3.5%-28.1 ± 3.5%22.2 ± 8.8%--23.9 ± 4.0%-104.7
F6-STheoretical--50.0%50.0%----
AGOC #1--46.4% (39)53.6% (44)----84
AGOC #2--50.0% (49)50.0% (49)----98
AGOC #3--54.0% (68)46.0% (58)----126
AGOC #4--53.8% (50)46.2% (43)----93
AGOC #5--51.3% (59)48.7% (56)----115
AGOC #6--57.0% (49)43.0% (37)----86
Mean--52.1 ± 3.7%47.9 ± 3.7%----100.3
F6Theoretical--25.0%25.0%--50.0%--
AGOC #1--20.3% (23)24.8% (28)--54.9% (62)-113
AGOC #2--21.5% (23)35.5% (38)--43.0% (46)-117
AGOC #3--22.9% (27)22.9% (27)--54.2% (64)-118
AGOC #4--22.0% (29)22.7% (30)--55.3% (73)-132
AGOC #5--17.5% (18)31.1% (32)--51.4% (53)-103
AGOC #6--19.8% (22)24.3% (27)--55.9% (62)-111
Mean--20.7 ± 1.9%26.9 ± 5.2%--52.4 ± 4.9%-115.7
F7-OTheoretical--100%------
AGOC #1--100% (94)-----94
AGOC #2--100% (50)-----50
AGOC #3--100% (49)-----49
AGOC #4--100% (79)-----79
AGOC #5--100% (79)-----79
AGOC #6--100% (63)-----63
Mean--100 ± 0%-----69.0
F7-CTheoretical---100%-----
AGOC #1---100% (101)----101
AGOC #2---100% (105)----105
AGOC #3---100% (89)----89
AGOC #4---100% (54)----54
AGOC #5---100% (74)----74
AGOC #6---100% (64)----64
Mean---100 ± 0%----81.2
  1. *In the AGOC #4 subline, incomplete recombination occurred in the F4 (mCe; mO-mC) double hemizygous generation, as we obtained several F5 individuals that still carried both transformation markers (7.0% in total). We continued the mating procedure with the F5 (mO) and (mC) post-recombination hemizygous progeny.

Throughout all generations, the subline-specific scores matched the expectations. No significant differences between the respective arithmetic means and the theoretical Mendelian ratios were found. Importantly, all expected phenotypes, and thus all expected genotypes, were found in all generations and consequently, F7 (mO/mO) as well as (mC/mC) homozygotes were obtained for all six AGOC sublines. A description of the generations and their characteristics is found in Supplementary file 4.

Two controls were performed with the AGOC #5 and #6 sublines to confirm proper function of the AGOC vector concept: The F3 to F7 crossing procedure was successfully conducted with (i) swapped genders (Figure 3—figure supplement 2A, Supplementary file 5) and (ii) an alternative helper subline (Figure 3—figure supplement 2B, Supplementary file 5), ICE{HSP68’NLS-Cre} #2, which carries the same Cre recombinase-expressing cassette as the #1 subline, but at a different genomic location.

Systematic creation of functional homozygous AGOC lines and sublines

Based on pAGOC, we developed the intermediate pAGOC{#P’#O(LA)-mEmerald} vector (Figure 1—figure supplement 2), which contains an open-reading frame for mEmerald-labeled (Shaner et al., 2005) Lifeact, a small and universal peptide tag derived from Saccharomyces cerevisiae that binds to filamentous actin (Riedl et al., 2008). With this vector, three transformation-ready derivates were created that allow expression of mEmerald-labeled Lifeact under control of either the tubulin alpha 1-like protein (Siebert et al., 2008), the zerknüllt 1 (van der Zee et al., 2005; ; Sharma et al., 2013; Panfilio et al., 2013; Hilbrant et al., 2016) or the actin-related protein 5 promoter. Additionally, we developed two more derivates that contain expression cassettes for either mEmerald-labeled beta-galactoside alpha-2,6-sialyltransferase 1 or histone H2B under control of the tubulin alpha 1-like protein promoter. With these vectors, we created five functional lines with one, three, two, three and four sublines, respectively (that is, 13 in total), which are primarily designed for fluorescence live imaging. Twelve of these sublines went through the procedure successfully, only the AGOC{ATub’H2B-mEmerald} #4 subline turned out to be heterozygous/homozygous sterile (Supplementary file 6 for the mEmerald-labeled Lifeact-expressing sublines and Supplementary file 7 for the mEmerald-labeled beta-galactoside alpha-2,6-sialyltransferase 1- and histone H2B-expressing sublines).

Fluorescence live imaging of selected functional homozygous AGOC sublines

We performed long-term fluorescence live imaging of the embryonic development (Strobl and Stelzer, 2016) with three of the functional (mC/mC) homozygous sublines. We used a digital scanned laser light-sheet-based fluorescence microscope (Keller et al., 2008; Keller and Stelzer, 2010) in conjunction with previously published sample preparation protocols for Tribolium (Strobl and Stelzer, 2014; Strobl et al., 2015Strobl et al., 2017a). The AGOC{Zen1’#O(LA)-mEmerald} #2 subline allows the characterization of actin dynamics within certain extra-embryonic membrane progenitor cells during gastrulation, visualizing the actomyosin cable that closes the serosa window (Figure 4A and Video 1). The AGOC{ARP5’#O(LA)-mEmerald} #1 subline provides strong fluorescence signal in the brain and ventral nerve cord and moderate signal throughout the remaining embryonic tissue (Figure 4B and Video 2). In contrast, the AGOC{ARP5’#O(LA)-mEmerald} #2 subline provides uniform fluorescence intensity during gastrulation, germband elongation, germband retraction and dorsal closure (Figure 4C and Video 3).

Fluorescence live imaging of selected functional (mC/mC) homozygous AGOC sublines.

(A) An AGOC{Zen1’#O(LA)-mEmerald} #2 embryo during gastrulation. This subline permits the characterization of actin and actomyosin dynamics involved in serosa window closure (first row). It can also be used to describe the cytoskeleton rearrangement of the dorsal blastoderm cells (second row) and to analyze their change in appearance during differentiation to serosa cells (third row and enlarged images). (B) An AGOC{ARP5’#O(LA)-mEmerald} #1 embryo during germband retraction. In this subline, the brain and ventral nerve cord express mEmerald-labeled Lifeact on a high level, permitting the observation of neurulation. Enlarged images show the forming ganglia of the first and second thoracic segments. (C) Comparison of embryos from the AGOC{ARP5’#O(LA)-mEmerald} #1 and #2 sublines after dorsal closure. In contrast to the #2 subline, the fluorescence signal within the nervous system of the #1 subline is noticeably strong. ZP, Z maximum projection with image processing; ZA, Z maximum projection with intensity adjustment.

https://doi.org/10.7554/eLife.31677.016
Video 1
Long-term live imaging of a (mC/mC) homozygous Tribolium embryo from the AGOC{Zen1’#O(LA)-mEmerald} #2 subline.

Embryogenesis is shown along four directions from 00:00 hr to 24:00 hr with an interval of 00:30 hr between the time points. The video starts with the rearrangement of the blastoderm and ends during germband retraction. During gastrulation, the ventrally located serosa window is closed by a contracting actomyosin cable that separates the serosa and the amnion. Frame rate is five frames per second. ZP, Z maximum projection with image processing.

https://doi.org/10.7554/eLife.31677.017
Video 2
Long-term live imaging of a (mC/mC) homozygous Tribolium embryo from the AGOC{ARP5’#O(LA)-mEmerald} #1 subline.

Embryogenesis is shown along four directions from 00:00 hr to 96:00 hr with an interval of 00:30 hr between the time points. The video starts with the rearrangement of the blastoderm and ends after dorsal closure. This transgenic line exhibits strong fluorophore expression in the ventral nerve cord. Frame rate is five frames per second. ZA, Z maximum projection with image adjustment.

https://doi.org/10.7554/eLife.31677.018
Video 3
Long-term live imaging of a (mC/mC) homozygous Tribolium embryo from the AGOC{ARP5’#O(LA)-mEmerald} #2 subline.

Embryogenesis is shown along four directions from 00:00 hr to 120:00 hr with an interval of 00:30 hr between the time points. The video starts with the rearrangement of the blastoderm and ends after dorsal closure. In contrast to the #1 subline (Video 2), this subline does not exhibit strong fluorophore expression in the ventral nerve cord. Frame rate is five frames per second. ZA, maximum projection with image adjustment.

https://doi.org/10.7554/eLife.31677.019

Discussion

We explained the abstract genetic background of the AGOC vector concept and confirmed its straightforward applicability with Tribolium. The unique feature of our approach is that temporary ambiguities are avoided in any generation, since all genotypes are directly identified by specifically designed distinct phenotypes. Hence, AGOC-based workflows can be used to systematically create progeny with relevant genotypes, as exemplified in this study for the creation of homozygous lines. Consequently, our concept provides many advantages that apply not only to Tribolium but also to many other model organisms: (i) Our approach saves manpower. For example, genotyping 30 to 40 Tribolium adults with genetic assays takes about one afternoon (Strobl et al., 2017b), while processing the same number of individuals with a stereo microscope takes less than ten minutes. (ii) The concept does not require any further consumables. (iii) When genetic assays are used, the ‘slowest’ member of a group defines the earliest convenient time point for synchronized genotyping, while our concept also supports unsynchronized genotyping of single organisms. (iv) Our approach is non-invasive and thus favorable when invasive procedures are incompatible with the experimental workflow. It can be performed even when sufficient amounts of genomic DNA cannot be obtained without severely injuring or even sacrificing the individual. (v) The concept simplifies transgenic organism handling since genotypes are determined directly. Quick and reliable quantification, selection, mating and/or grouping of individuals can be performed during nearly all developmental stages. (vi) Our approach is less error-prone than genetic assays. In more than 300 independent instances, the progeny scores confirmed the phenotypically determined parental genotypes. (vii) Although homozygous transgenic lines can be systematically created with slightly less waiting time by using balancer chromosomes, a convenient number of balancer lines is only available for Drosophila (Ashburner, 1989). Furthermore, in the balancer-based approach, the insertion location has to be known, while our approach performs properly in random and semi-random insertion assays. (viii) Many special cases of transgenesis (four cases that occurred during our study are described within the Materials and methods section) can be explicitly identified and/or attended to. (ix) Specifically designed distinct phenotypes foster automation. For example, several approaches for the computer-controlled allocation of zebrafish embryos to 96-well plates have been suggested (Graf et al., 2011; Mandrell et al., 2012). Automation devices, equipped with a phenotype-adapted detection unit, in our case fluorescence, can be used to sort organisms with different genotypes according to their markers.

The functionality of the AGOC vector concept was confirmed with Tribolium, but due to the universality of the Cre-Lox system, it should work in all diploid model organisms. These include various insects, zebrafish, rodents, and even plants. For many insect species, modifying the basic architecture of the vector is not necessary. It has been shown that both the piggyBac transposon system and the 3×P3 promoter function properly in Drosophila melanogaster (Sarkar et al., 2006), its close relative Drosophila suzukii (Schetelig and Handler, 2013) and many other dipterans (Hediger et al., 2001; Warren et al., 2010; Caroti et al., 2015), including epidemiologically relevant mosquito species such as Aedes aegypti (Kokoza et al., 2001) and Aedes albopictus (Labbé et al., 2010). This also applies to multiple lepidopterans such as Bicyclus anynana (Marcus et al., 2004) and Bombyx mori (Thomas et al., 2002), other coleopterans (Kuwayama et al., 2006) as well as some hymenopterans, for example the honeybee Apis mellifera (Schulte et al., 2014). For several other dipteran species, such as the African malaria mosquito Anopheles gambiae (Grossman et al., 2001) as well as several tephritid (Handler and Harrell, 2001; Schetelig et al., 2009; Raphael et al., 2011) and calliphorid (Heinrich et al., 2002; Allen et al., 2004) species, the marker cassettes have to be modified, for example by replacing the artificial 3×P3 promoter with the Drosophila polyubiquitin promoter. If fluorescence-based markers interfere with the experimental workflow, pigmentation-based markers can be used. Eye pigmentation markers are available for Drosophila melanogaster (Adams and Sekelsky, 2002) and Tribolium castaneum (Lorenzen et al., 2002), but require the appropriate background strain. Another convenient and apparently universal option for insects are arylalkylamine-N-acetyl transferase-based markers, which lighten pigmentation throughout the cuticle and thus can be detected without microscopes (Osanai-Futahashi et al., 2012). Although the piggyBac transposon system works properly in zebrafish (Lobo et al., 2006) and the 3×P3 promoter is believed to work in a broad variety of animal species (Berghammer et al., 1999), it may be convenient to transit to the well-established Tol2 transposon system (Kawakami et al., 2000) and to replace the 3×P3 promoter in the marker cassettes with endogenous alternatives, for example the eye-specific cryaa or the muscle-specific 503unc promoter (Berger and Currie, 2013). For mouse, epidermal (Ikawa et al., 1995; Zhu et al., 2005) and eye-specific (Cornett et al., 2011) fluorescence-based as well as fur color-based (Zheng et al., 1999) markers have been established.

In this study, we used the AGOC vector concept in conjunction with transposon-mediated transgenesis to systematically create functional homozygous Tribolium lines that are primarily designed for fluorescence live imaging of embryonic development. However, our approach can also be used in insertional mutagenesis knock-out assays, independent of whether large-scale transposon-mediated remobilization with subsequent screening is performed (Trauner et al., 2009), or genes or genetic elements are specifically rendered inoperative, for example, by using genome engineering techniques such as CRISPR/Cas9, where AGOC-based transgenes can be integrated into targeted genomic locations via either homology-based repair or non-homologous end-joining (Gilles and Averof, 2014; Gilles et al., 2015).

Studies that utilize genetically manipulated organisms require researchers to rear their lines for many years. A certain numbers of individuals with known genotypes are required during this period, either to maintain the lines or to use them in experiments. This results in a total demand of hundreds to thousands of organisms. The AGOC vector concept supports well-designed experimental strategies in the following scenarios: (i) Transgenic lines that are, for example, specifically designed for fluorescence live imaging are easily maintained as homozygotes since continuous genotyping and/or curation are not necessary. However, the initial workload following transgenesis that is required to create homozygous lines can be very high and thus a limiting factor. As shown in this study, these efforts are significantly reduced by using the AGOC vector concept. (ii) In knock-out assays of genes that result in homozygous lethality, the respective lines are maintained as hemizygotes, which are usually viable and phenotypically inconspicuous. When two hemizygous organisms are mated, only one quarter of their progeny are homozygous for the knock-out, while half are hemizygous and one quarter resemble the wild type. Certain experimental approaches, for example, fluorescence live imaging or transcriptome/proteome analyses, require the researcher to commence with all descendants and to select the homozygous knock-out individuals as soon as discrimination is possible, that is, when the phenotype manifests or when biological material for genetic assays can be obtained. By using our approach with appropriate markers, a preselection can be performed. This narrows the efforts down to relevant individuals and appropriate controls, for example, down to about one quarter of the currently required number. The AGOC vector concept, broadly adapted to established and emerging model organisms, contributes significantly to ethically motivated endeavors to minimize the number of wasted animals.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Tribolium castaneum)Tubulin alpha 1-like protein (ATub)PMID: 18625397Gene ID: 656649-
Gene (T. castaneum)Heat shock protein 68b (HSP68)PMID: 22890852Gene ID: 100142517-
Gene (T. castaneum)Zerknüllt 1 (Zen1)This paperGene ID: 641533The Tribolium Zen1 promoter
was not cloned previously
Gene (T. castaneum)Actin-related protein 5 (ARP5)This paperGene ID: 655949The Tribolium ARP5 promoter
was not cloned previously
Gene (T. castaneum)Histone H2B (H2B)This paperGene ID: 661713The Tribolium H2B open-reading
frame was not cloned previously
Gene (T. castaneum)Beta-galactoside alpha-2,6-
sialyltransferase 1 (SiaTr)
This paperGene ID: 657186The Tribolium SiaTr open-reading
frame was not cloned previously
Strain, strain background
(T. castaneum)
Plain-White-As-Snow
(PWAS) background strain
PMID: 25555987-New hybrid: pearl background strain that
also carries the light ocular diaphragm mutation
Recombinant DNA reagentpUC57[AGOC]This paper-Gene synthesis (Genewiz)
Recombinant DNA reagentpGS[#P’#O(LA)-mEmerald]This paper-Gene synthesis (Invitrogen)
Recombinant DNA reagentpGS[ACOS]This paper-Gene synthesis (Invitrogen)
Recombinant DNA reagentpBSII-IFP-CDSPMID: 19716359-Kind gift from Malcom Fraser (University
of Notre Dame, Indiana, United States)
Recombinant DNA reagentpTriEx-HTNCPMID: 11904364Addgene ID: 13763Ordered from Addgene.
Recombinant DNA reagentpGEM-T EasyPromegaCatalog No: A1360-

Tribolium castaneum strains and rearing

For this study, a T. castaneum (NCBITaxon:7070) double mutant background strain was created, which carries the pearl (Grubbs et al., 2015) and light ocular diaphragm (Mocelin and Stuart, 1996) mutations that result in completely unpigmented eyes. This strain was called Plain-White-As-Snow (PWAS) and used as a donor for genomic DNA and messenger RNA as well as for the creation of transgenic lines. Cultures were kept in groups of 150–500 individuals on growth medium (full grain wheat flour (113061006, Demeter) supplemented with 5% (wt/wt) inactive dry yeast (62–106, Flystuff) in 1 l glass bottles in a 12:00 hr light/12:00 hr darkness cycle at 25°C and 70% relative humidity (DR-36VL, Percival Scientific).

Tribolium genomic DNA/mRNA extraction and complementary DNA synthesis

Approximately 20 PWAS adults (40 mg) were starved for 24 hr before genomic DNA was extracted with the Blood and Tissue Kit (69504, Qiagen) according to the manufacturer’s instructions. From approximately 10 adults (20 mg), messenger RNA was extracted with TRIzol Reagent (15596026, Thermo Fisher Scientific) and complementary DNA was transcribed with the Superscript III reverse transcriptase (12080093, Thermo Fisher Scientific) using random hexamer primers.

Experimental design

For germline transformation, the piggyBac transposon system (Handler, 2002) was chosen, which is highly active in Tribolium. This study utilized a set of vectors based on in silico design and de novo synthesis. This section explains the architecture of the three most important vectors in general, while the detailed molecular biological procedure is explained within the following sections. All intermediate and transformation-ready vectors used in this study are based on pAVOIAF{#1–#2–#3–#4} (Figure 1—figure supplement 3). Between the unique AatII and PciI sites, this vector carries a transposon cassette which consists of (i) the piggyBac 3’ terminal repeat, (ii) a four-slot (#1 to #4) cloning site and (iii) the 5’ piggyBac terminal repeat. The repeats have the minimal length (235 and 310 bp, respectively) necessary for efficient transposition (Li et al., 2005). The four-slot cloning site consists of four restriction enzyme site pairs, XmaI/SpeI for #1, HindIII/XbaI for #2, XhoI/NheI for #3 and AflII/AvrI for #4. The pairs are separated by 18 bp Phe-Arg-Glu-Asp-Asp-Tyr (FREDDY) spacers. For convenience, PmeI sites were placed at upstream and downstream of the four-slot cloning site as well as between the restriction enzyme site pairs.

In #3 and #4 of pAVOIAF{#1–#2–#3–#4}, mOrange- and mCherry-based eye-specific transformation markers (mO and mC, respectively) were inserted (in reverse orientation) that consist of (i) the artificial 3×P3 promoter (Berghammer et al., 1999), (ii) the codon-optimized open-reading frames for the respective fluorescent protein, that is, mOrange2 (Shaner et al., 2008) or mCherry (Shaner et al., 2004) and (iii) the SV40 poly(A) (van den Hoff et al., 1993). Both markers are flanked by incompatible Lox sites (the spacer is underlined, deviations are marked bold): upstream by a LoxP (5’-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3’) and downstream by a LoxN (5’-ATAACTTCGTATAGTATACCTTATACGAAGTTAT-3’) site. For convenience, the resulting vector was termed pAGOC (Figure 1—figure supplement 1).

In #2 of pAGOC, a modular fluorescent protein expression cassette was inserted, which consists of (i) a two-slot cloning site composed of a promoter (#P) and an open-reading frame (#O) slot, (ii) a 9 bp Ala-Ala-Ala linker, (iii) the codon-optimized mEmerald open-reading frame (Tsien, 1998) and (iv) an elongated variant of the SV40 poly(A) (van den Hoff et al., 1993). The #P slot can be accessed by the AscI/FseI site pair, or alternatively scarlessly by the double BtgZI site pair. The #O slot carries the open-reading frame for the Saccharomyces cerevisiae Lifeact peptide tag (Riedl et al., 2008) per default and can be accessed by the FseI/NotI site pair. The mEmerald open-reading frame can be accessed by the NotI/SbfI site pair. For convenience, the resulting vector was termed pAGOC{#P’#O(LA)-mEmerald} (Figure 1—figure supplement 2). Any exogenous or endogenous promoter can be inserted in the #P slot to generate the spatiotemporal activity pattern of choice. For the #O slot, the Lifeact open-reading frame can be replaced with any open-reading frame to change the subcellular localization of the fluorescent protein. In the default configuration, the Lifeact peptide tag will guide mEmerald to the actin cytoskeleton.

Molecular biology

In this study, 25 vectors (Figure 1—figure supplement 4 and Supplementary file 8) plus the commercial subcloning vector pGEM-T Easy (A1360, Promega) were used. Three were ordered as gene synthesis plasmids, two were previously published and obtained from the respective laboratories or from Addgene, six are library vectors, while the remaining 13 vectors are derivates. For all PCRs, Phusion High Fidelity DNA polymerase (M0530L, New England BioLabs) was used, and T4 DNA ligase (M0202L, New England BioLabs or provided with the pGEM-T Easy vector) for all ligations. Cloning primers are listed in Supplementary file 9.

Molecular biology: the promoter and open-reading frame library vectors

The respective promoter and open-reading frame sequences were amplified from genomic or complementary DNA by using the appropriate extraction PCR primer pairs (C1 for tubulin alpha 1-like protein (ATub’), C2 for zerknüllt 1 (Zen1’), C3 for actin-related protein 5 (ARP5’) and C4 for heat shock protein 68b (HSP68’) as well as C5 for beta-galactoside alpha-2,6-sialyltransferase 1 transcription variant X1 (’SiaTr) and C5 for histone H2B (’H2B)). Amplification was followed by A-tailing using the Recombinant Taq DNA polymerase (10342020, Thermo Fisher Scientific) and ligation into pGEM-T Easy. The resulting vectors were termed pTC-ATub’-GEM-T Easy, pTC-Zen1’-GEM-T Easy, pTC-ARP5’-GEM-T Easy, pTC-HSP68’-GEM-T Easy, pTC-’SiaTr-GEM-T Easy and pTC-’H2B-GEM-T Easy. To create the hybrid promoter/open-reading frame library vectors, the sequences were amplified from the library vectors or pTriEx-HTNC (Peitz et al., 2002) with the respective fusion PCR primer pairs (either C7 for HSP68’ / ’NLS-Cre, or C8 for ATub’ / ’H2B) and fused in a secondary PCR reaction using both PCR products as a template and the promoter forward primer (C7-1 or C8-1, respectively) and the open-reading frame reverse primer (C7-4 or C8-4, respectively). The primer pairs introduce upstream an AscI and downstream a NotI site or upstream a NheI and downstream a XhoI site, respectively. The fusion PCR products were inserted into pGEM-T Easy as described above. The resulting vectors were termed pTC-ATub’H2B-GEM-T Easy and pTC-HSP68’NLS-Cre-GEM-T Easy.

Molecular biology: the pUC[AGOC] and pAGOC vectors

A hybrid sequence, consisting of (i) the transposon cassette as well as (ii) mO and mC and their flanking Lox sites in #3 and #4 as described above, was de novo synthetized and inserted into the unique NdeI and PstI sites of pUC57-Kan (GeneBank accession number JF826242.2). The resulting vector was termed pUC57[AGOC]. The insert was PCR amplified with primer pair C9, which introduced upstream an AatII and downstream a PciI site. The PCR product and pUC57[AGOC] were digested accordingly, and the insert was reintegrated into the vector, removing 629 functionless bp. The resulting vector was termed pAGOC and used (i) as a transformation-ready vector for germline transformation, and (ii) as an intermediate vector for further cloning operations.

Molecular biology: the pGS[#P’#O(LA)-mEmerald] and pAGOC{#P’#O(LA)-mEmerald} vectors

A hybrid sequence, consisting of (i) a HindIII site, (ii) the modular fluorescent protein expression cassette as described above and (iii) a XbaI site, was de novo synthetized and inserted into the unique SfiI site of pMK-RQ (Thermo Fisher Scientific). The resulting vector was termed pGS[#P’#O(LA)-mEmerald]. The insert was excised from the backbone with HindIII/XbaI and inserted into #3 of the pAGOC vector. The resulting vector was termed pAGOC{#P’#O(LA)-mEmerald} and used as an intermediate vector for further cloning operations.

Molecular biology: the pAGOC{ATub’#O(LA)-mEmerald}, pAGOC{Zen1’#O(LA)-mEmerald} and pAGOC{ARP5’#O(LA)-mEmerald} vectors

The respective promoter sequences were amplified from the library vectors with the respective primer pairs (C10 for ATub’, C11 for Zen1’ and C12 for ARP5’), which introduced upstream an AscI and downstream a BsmBI (ATub’) or BsaI (Zen1’ and ARP5’) site. The PCR products were digested accordingly, and the pAGOC{#P’#O(LA)-mEmerald} vector was digested with BtgZI, which led to compatible overhangs and allowed scarless insertion of the promoter sequences into #P. The resulting vectors were termed pAGOC{ATub’#O(LA)-mEmerald}, pAGOC{Zen1’#O(LA)-mEmerald} and pAGOC{ARP5’#O(LA)-mEmerald} and were used for germline transformation.

Molecular biology: the pAGOC{#P’SiaTr-mEmerald} and pAGOC{ATub’SiaTr-mEmerald} vectors

The ’SiaTr open-reading frame sequence was amplified from the pTC-’SiaTr-GEM-T Easy vector with primer pair C13, which introduced upstream an FseI and downstream a NotI site. The PCR product and the pAGOC{#P’#O(LA)-mEmerald} vector were digested accordingly and the insert was inserted into #O of the vector. The resulting vector was termed pAGOC{#P’SiaTr-mEmerald} and used as an intermediate vector for further cloning operations. The tubulin alpha 1-like protein promoter sequence was amplified from the pTC-ATub’-GEM-T Easy vector with primer pair C10, which introduced upstream an AscI and downstream a BsmBI site. The PCR product was digested accordingly, and the pAGOC{#P’SiaTr-mEmerald} vector was digested with BtgZI, which led to compatible overhangs and allowed scarless insertion of the promoter sequence into #P of the intermediate vector. The resulting vector was termed pAGOC{ATub’SiaTr-mEmerald} and used for germline transformation.

Molecular biology: the pAGOC{ATub’H2B-mEmerald} vector

The ATub’H2B promoter/open-reading frame sequence was excised from pTC-ATub’H2B-GEM-T Easy with AscI and NotI and inserted into #P’#O of the accordingly digested pAGOC{#P’#O(LA)-mEmerald} vector. The resulting vector was termed pAGOC{ATub’H2B-mEmerald} and used for germline transformation.

Molecular biology: the pAVOIAF{#1–#2–HSP68’NLS-Cre–mC}, pGS[ACOS] and pICE{HSP68’NLS-Cre} vectors

The HSP68’NLS-Cre recombinase promoter/open-reading frame sequence was excised from pTC-HSP68’H2B-GEM-T Easy with NheI and XhoI and inserted (in reverse orientation) into #3 of the accordingly digested pAGOC vector, replacing mO and the flanking Lox sites. The resulting vector was termed pAVOIAF{#1–#2–HSP68’NLS-Cre–mC} and used as an intermediate vector for further cloning operations. A hybrid sequence, which consists (beside other elements) of the mCerulean-based eye-specific transformation marker (mCe) that is composed of (i) the artificial 3×P3 promoter, (ii) the codon-optimized open-reading frame for mCerulean2 (Markwardt et al., 2011) and (iii) the SV40 poly(A), was de novo synthetized and inserted into the unique SfiI site of pMK-RQ (Thermo Fisher Scientific). The resulting vector was termed pGS[ACOS]. Next, mCe was amplified with primer pair C14, which introduced upstream an AflII and downstream an AvrII site. The PCR product was digested accordingly and inserted into #4 of pAVOIAF{#1–#2–HSP68’NLS-Cre–mC}, replacing mC and the flanking Lox sites. The resulting vector was termed pICE{HSP68’NLS-Cre} and used for germline transformation to create the Cre recombinase-expressing helper lines.

Molecular biology: the pATub’piggyBac vector

The ATub promoter and the piggyBac open-reading frame fragment were amplified from pTC-ATub’-GEM-T Easy and pBSII-IFP-ORF (Yoshida et al., 2009) with the C15 primer pairs and fused together in a secondary PCR reaction using both PCR products as a template as well as the promoter forward primer (C15-1) and the open-reading frame reverse primer (C15-4). The primers introduced upstream a SalI and downstream a BglII site, respectively. The fusion PCR product was digested and then reintegrated into the accordingly digested pBSII-IFP-ORF vector. The resulting vector was termed pATub’piggyBac and used as the transposase-expressing helper vector during germline transformation.

Germline transformation

Approximately 500 F0 PWAS adults were incubated on 405 fine wheat flour (113061036, Demeter, Darmstadt, Germany) supplemented with 5% (wt/wt) inactive dry yeast (62–106, Flystuff, San Diego, CA) at 25°C and 70% relative humidity in light for 2 hr. After the incubation period, the adults were removed and the embryos (around 700 to 900) were extracted from the flour and incubated another hour as stated above. Next, the embryos were briefly washed in 10% (vol/vol) sodium hypochlorite (425044–250 ML, Sigma Adlrich) in autoclaved tap water for 10 s, stored in autoclaved tap water and lined up on microscopy slides within the next hour. The embryos were injected with a mixture of 500 ng/µl transformation-ready vector and 400 ng/µl pATub’piggyBac in injection buffer (5 mM KCl, 1 mM KH2PO4 in ddH2O, pH 8.8). For injection, a microinjector (FemtoJet, Eppendorf) and 0.7 µm outer diameter capillaries (Femtotips II, Eppendorf) with an injection pressure of 400–800 hPa were used. After injection, the microscopy slides with embryos were placed on a 5 mm high 1% (wt/vol) broad range agarose (T846.3, Carl Roth) in tap water ‘platform’ within Petri dishes and incubated at 32°C. After 3 days, hatched larvae, that is, F1 potential mosaics, were collected and raised individually in single wells of 24-well plates as described above. Germline transformation resulted in a total of seven lines with 21 sublines, which are summarized in Supplementary file 10.

Mating procedure, insert number determination cross and homozygous viability cross

All crossings were performed with single female-male pairs in small glass vials filled with 1.5 g or 2.5 g (F4 cross) of growth medium. Progeny were placed individually in wells of 24-well plates and scored for the presence of markers during pupal or adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Supplementary file 11). For each pair, images in the reflected light and fluorescence channels were taken in parallel with appropriate controls. The mating procedure is described within the results section. A one-sample/two tailed Student’s t-test was performed to determine whether the arithmetic means differ significantly from the theoretical Mendelian ratios. Insert numbers were determined by mating F2 hemizygotes with wild types and scoring the progeny, whereas a transgene distribution of 60% or less was interpreted as a single insertion. Homozygous viability was determined by mating two F3 hemizygotes and scoring the progeny, whereas a transgene distribution of 70% or more was interpreted as a homozygous viable line.

The AGOC vector concept in special cases of transgenesis

During the experimental validation of the AGOC vector concept, four special cases of transgenesis occurred and were attended to as follows: (i) The homozygous viability crosses indicated that the transgenes of the proof-of-principle AGOC #3, the functional AGOC{ATub’#O(LA)-mEmerald} #1 and the functional AGOC{ATub’H2B-mEmerald} #4 sublines are heterozygous/homozygous lethal (Supplementary file 2). However, F6 (mO/mC) heterozygotes were obtained for all three lines and the mating procedure could be performed successfully with the AGOC #3 and AGOC{ATub’#O(LA)-mEmerald} #1 sublines (Table 1, Supplementary file 6 and Supplementary file 7). The mating procedure only aborted for the AGOC{ATub’#O(LA)-mEmerald} #4 subline, because the F6 (mO/mC) heterozygotes were sterile. Since the transgene of the proof-of-principle AGOC #3 subline might interfere with the sialin-like gene due to its insertion location (Supplementary file 3), and since both functional sublines mentioned above display a strong green fluorescence signal, hemizygous individuals of those three sublines are believed to be exposed to high stress levels, and that these levels are even higher in homozygotes. Thus, these transgenic sublines are believed to be essentially viable, but a certain percentage of the descendants fail to develop properly, which results in biased progeny ratios in the homozygous viability cross. (ii) In contrast to heterozygous/homozygous lethality, heterozygous/homozygous sterility cannot be estimated from the homozygous viability crosses. The AGOC{ATub’H2B-mEmerald} #4 subline was assumed to be heterozygous/homozygous lethal (Supplementary file 2), but mating a F5 (mO) post-recombination hemizygous female with a F5 (mC) post-recombination hemizygous male sibling resulted in F6 (mO/mC) heterozygotes. However, by mating F6 (mO/mC) heterozygous females with genotypically identical F6 males, no progeny was obtained (n = 12). Further, crossing F6 (mO/mC) heterozygous females and wild-type males did not result in any progeny (n = 12). To confirm sterility of both genders, F6 (mO/mC) heterozygous males were mated with wild-type females, which also did not result in any progeny (n = 8). (iii) The AGOC{Zen1’#O(LA)-mEmerald} #2 subline carries the transgene not on one of the nine autosomes, but on the X allosome. Thus, a slightly modified mating procedure was necessary to obtain F7 (mO/mO) and (mC/mC) homozygotes (Figure 2—figure supplement 2). (iv) The piggyBac transposon system is highly efficient in Tribolium, which results to a certain degree also from the 4 bp TTAA target sequence. However, due to this very short length of the targeting sequence, a certain probability for nested insertions is given, that is, the transgene inserts into another transformation vector, since these vectors also carry multiple TTAA target sequences. In the AGOC{Zen1’#O(LA)-mEmerald} #3 subline, an insertion of the transgene into the backbone of another vector occurred, and the nested transgene was subsequently inserted into the genome, as revealed by sequencing of the insertion junction (Supplementary file 3). This rare and undesired case is ‘corrected’ during the mating procedure of the AGOC vector concept, since within the F4 (mCe; mO-mC) double hemizygous generation, the Cre recombinase excises nearly one ‘stitched equivalent’ of the initial transformation vector from the genome. The F5 (mO) and (mC) hemizygous progeny then carries only one, but complete copy of the transgene.

One of the most obvious special cases of transgenesis, heterozygous/homozygous lethality, did not occur. However, the AGOC vector concept would allow the determination of this special case with a high degree of certainty. When F5 (mO) post-recombination hemizygous females are mated with F5 (mC) post-recombination hemizygous male siblings and no F6 (mO/mC) heterozygotes are obtained, it can be assumed that the transgene is heterozygous/homozygous lethal. Multiple other transgenesis special cases are possible (e.g. female- or male-only heterozygous/homozygous lethality or sterility, nested insertions of transgenes into transgenes, multiple inserts in close proximity on the same chromosome), but are not discussed here due to the very low probability of their occurrence.

Determination of insertion junction via inverse PCR

To determine the insertion junction of the piggyBac-based transgene, the inverse PCR approach was chosen (Triglia et al., 1988; Ochman et al., 1988). All inverse PCR primers are listed in Supplementary file 9. At first, inverse PCR was performed for the junction at the 5’ piggyBac terminal repeat with the I-5’ primer pair with up to eight different restriction enzymes, and if unsuccessful, also at the 3’ piggyBac terminal repeat with the I-3’ primer pair with up to six different restriction enzymes. PCR products were extracted from the gel, A-tailed, ligated into pGEM-T Easy and sequenced. For each successful inverse PCR, a control PCR at the respective other side was performed. For the control PCR, location-specific primers were used to perform a standard PCR. Inverse PCR was successful for 10 out of 19 lines, for 4 out of 6 proof-of-principle lines and 6 out of 13 functional lines. The sequencing results were aligned to the Tribolium genome (Richards et al., 2008) via the BeetleBase (Wang et al., 2007; Kim et al., 2010) (RRID:SCR_001955) BLAST. Insertion junctions are listed in Supplementary file 3.

Light-sheet-based fluorescence microscopy

Long-term live imaging was performed with digitally scanned laser light-sheet-based fluorescence microscopy (DSLM, LSFM) (Keller et al., 2008; Keller and Stelzer, 2010) as described previously for Tribolium (Strobl and Stelzer, 2014; Strobl et al., 2015). In brief, embryo collection was performed with the F7+ continuative (mC/mC) homozygous lines for 1 hr at 25°C, and embryos were incubated for 15 hr at 25°C. Sample preparation took approximately 1 hr at room temperature (23 ± 1°C), so that embryos were at the beginning of gastrulation. Embryos were recorded along four pair-wise orthogonal directions, that is, in the orientations 0°, 90°, 180° and 270°, with an interval of 30 min. All shown embryos survived the imaging procedure, developed to healthy and fertile adults, and when mated with wild types, produced only transgenic progeny that were also fertile. Metadata for the three datasets is provided in Supplementary file 12.

References

  1. 1
  2. 2
  3. 3
    Drosophila: A Laboratory Handbook
    1. M Ashburner
    (1989)
    Plainview NY: Cold Spring Harbor Laboratory Press.
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
    Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase
    1. MD Lorenzen
    2. SJ Brown
    3. RE Denell
    4. RW Beeman
    (2002)
    Genetics 160:225–234.
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
    Genetic applications of an inverse polymerase chain reaction
    1. H Ochman
    2. AS Gerber
    3. DL Hartl
    (1988)
    Genetics 120:621–623.
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
    The genome of the model beetle and pest Tribolium castaneum
    1. S Richards
    2. RA Gibbs
    3. GM Weinstock
    4. SJ Brown
    5. R Denell
    6. RW Beeman
    7. R Gibbs
    8. RW Beeman
    9. SJ Brown
    10. G Bucher
    11. M Friedrich
    12. CJ Grimmelikhuijzen
    13. M Klingler
    14. M Lorenzen
    15. S Richards
    16. S Roth
    17. R Schröder
    18. D Tautz
    19. EM Zdobnov
    20. D Muzny
    21. RA Gibbs
    22. GM Weinstock
    23. T Attaway
    24. S Bell
    25. CJ Buhay
    26. MN Chandrabose
    27. D Chavez
    28. KP Clerk-Blankenburg
    29. A Cree
    30. M Dao
    31. C Davis
    32. J Chacko
    33. H Dinh
    34. S Dugan-Rocha
    35. G Fowler
    36. TT Garner
    37. J Garnes
    38. A Gnirke
    39. A Hawes
    40. J Hernandez
    41. S Hines
    42. M Holder
    43. J Hume
    44. SN Jhangiani
    45. V Joshi
    46. ZM Khan
    47. L Jackson
    48. C Kovar
    49. A Kowis
    50. S Lee
    51. LR Lewis
    52. J Margolis
    53. M Morgan
    54. LV Nazareth
    55. N Nguyen
    56. G Okwuonu
    57. D Parker
    58. S Richards
    59. SJ Ruiz
    60. J Santibanez
    61. J Savard
    62. SE Scherer
    63. B Schneider
    64. E Sodergren
    65. D Tautz
    66. S Vattahil
    67. D Villasana
    68. CS White
    69. R Wright
    70. Y Park
    71. RW Beeman
    72. J Lord
    73. B Oppert
    74. M Lorenzen
    75. S Brown
    76. L Wang
    77. J Savard
    78. D Tautz
    79. S Richards
    80. G Weinstock
    81. RA Gibbs
    82. Y Liu
    83. K Worley
    84. G Weinstock
    85. CG Elsik
    86. JT Reese
    87. E Elhaik
    88. G Landan
    89. D Graur
    90. P Arensburger
    91. P Atkinson
    92. RW Beeman
    93. J Beidler
    94. SJ Brown
    95. JP Demuth
    96. DW Drury
    97. YZ Du
    98. H Fujiwara
    99. M Lorenzen
    100. V Maselli
    101. M Osanai
    102. Y Park
    103. HM Robertson
    104. Z Tu
    105. JJ Wang
    106. S Wang
    107. S Richards
    108. H Song
    109. L Zhang
    110. E Sodergren
    111. D Werner
    112. M Stanke
    113. B Morgenstern
    114. V Solovyev
    115. P Kosarev
    116. G Brown
    117. HC Chen
    118. O Ermolaeva
    119. W Hlavina
    120. Y Kapustin
    121. B Kiryutin
    122. P Kitts
    123. D Maglott
    124. K Pruitt
    125. V Sapojnikov
    126. A Souvorov
    127. AJ Mackey
    128. RM Waterhouse
    129. S Wyder
    130. EM Zdobnov
    131. EM Zdobnov
    132. S Wyder
    133. EV Kriventseva
    134. T Kadowaki
    135. P Bork
    136. M Aranda
    137. R Bao
    138. A Beermann
    139. N Berns
    140. R Bolognesi
    141. F Bonneton
    142. D Bopp
    143. SJ Brown
    144. G Bucher
    145. T Butts
    146. A Chaumot
    147. RE Denell
    148. DE Ferrier
    149. M Friedrich
    150. CM Gordon
    151. M Jindra
    152. M Klingler
    153. Q Lan
    154. HM Lattorff
    155. V Laudet
    156. C von Levetsow
    157. Z Liu
    158. R Lutz
    159. JA Lynch
    160. RN da Fonseca
    161. N Posnien
    162. R Reuter
    163. S Roth
    164. J Savard
    165. JB Schinko
    166. C Schmitt
    167. M Schoppmeier
    168. R Schröder
    169. TD Shippy
    170. F Simonnet
    171. H Marques-Souza
    172. D Tautz
    173. Y Tomoyasu
    174. J Trauner
    175. M Van der Zee
    176. M Vervoort
    177. N Wittkopp
    178. EA Wimmer
    179. X Yang
    180. AK Jones
    181. DB Sattelle
    182. PR Ebert
    183. D Nelson
    184. JG Scott
    185. RW Beeman
    186. S Muthukrishnan
    187. KJ Kramer
    188. Y Arakane
    189. RW Beeman
    190. Q Zhu
    191. D Hogenkamp
    192. R Dixit
    193. B Oppert
    194. H Jiang
    195. Z Zou
    196. J Marshall
    197. E Elpidina
    198. K Vinokurov
    199. C Oppert
    200. Z Zou
    201. J Evans
    202. Z Lu
    203. P Zhao
    204. N Sumathipala
    205. B Altincicek
    206. A Vilcinskas
    207. M Williams
    208. D Hultmark
    209. C Hetru
    210. H Jiang
    211. CJ Grimmelikhuijzen
    212. F Hauser
    213. G Cazzamali
    214. M Williamson
    215. Y Park
    216. B Li
    217. Y Tanaka
    218. R Predel
    219. S Neupert
    220. J Schachtner
    221. P Verleyen
    222. F Raible
    223. P Bork
    224. M Friedrich
    225. KK Walden
    226. HM Robertson
    227. S Angeli
    228. S Forêt
    229. G Bucher
    230. S Schuetz
    231. R Maleszka
    232. EA Wimmer
    233. RW Beeman
    234. M Lorenzen
    235. Y Tomoyasu
    236. SC Miller
    237. D Grossmann
    238. G Bucher
    239. Tribolium Genome Sequencing Consortium
    (2008)
    Nature 452:949–955.
    https://doi.org/10.1038/nature06784
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
    Light sheet-based fluorescence microscopy of living or fixed and stained Tribolium castaneum embryos
    1. F Strobl
    2. S Klees
    3. EHK Stelzer
    (2017)
    Journal of Visualized Experiments, 10.3791/55629, 28518097.
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73

Decision letter

  1. Bruce Edgar
    Reviewing Editor; University of Utah, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines" for consideration by eLife. Your article has been favorably evaluated by K VijayRaghavan (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal her identity: Susan Brown (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

I have attached the reviews in full below, with the hope that you will be able to address essentially all of the requested revisions. Overall, the reviewers appreciated the content of the paper, but felt that it needs significant revision to make it clearer, more concise, and more accessible to a broad readership. The revision thus should have simpler figures, less supplementary data, and more concise Results and Discussion sections. I think it will also be important to revise the rationale, putting your method's strengths and weaknesses better in context with existing methods for selecting and working with mutants and transgenics. Reviewer #3 had some important contents in this area. I look forward to seeing your revision.

Reviewer #1:

In this paper, Strobl et al. present a germline transformation protocol using a novel vector they've developed, that streamlines the process of making a transgene insert into the genome homozygous. The concept is simple yet novel and applicable, with modifications, to a wide range of animal genetic models that lack other means of easily homozygosing transgenes, such as by the use of balancers or targeted insertion sites. The authors demonstrate their technique using vectors they've constructed and fluorescent eye markers in the flour beetle, Tribolium. The technique works, and would appear to be a useful one for this beetle and other model genetic systems that lack advanced genetic tools. Overall the paper is well put together; it's complete and easy to understand and includes the tests needed to validate the method presented. However the paper's methods and figures sections are much longer and more detailed than they need to be, so I suggest simplifying these to make the whole more accessible to the interested reader. Specific comments follow below:

1) Subsection “Systematic creation of homozygous lines”, first paragraph. To put things in perspective, this would take 3 generations in Drosophila using balancers, so a 4 generation procedure in Tribolium is not revolutionary, but rather an incremental advance.

2) The complete DNA sequences of the vectors should be made available as supplemental data for this paper. This is quite important to make the method accessible to the scientific community.

3) Would it not make sense to invert one of the marker genes in the vector to prevent read-through? Please comment.

4) In Figure 2, why is the first box F3 rather than F1 or F2? Please comment. Why is there a dotted line connecting rows 3 and 4, rather than solid?

5) Figures 3 and 4 are much more complex than necessary, and they didn't reproduce well. Please simplify these two figures.

6) The text in Table 1 is too small to read. Could this be presented in a more intuitive form?

7) The supplementary data are longer and more complex than necessary. Please include only what is necessary to convey the method to the reader.

8) In the Discussion, please include a list of the organisms in which this technique might be useful, and discuss how the vector would have to be modified to be used in these other organisms. Discussing these issues is important to ensure adoption of this method by the broader research community.

Reviewer #2:

The authors describe important technical improvements to transgenic vector constructs that allow efficient production of homozygous transgenic strains. Their concept is applicable to any system in which one wishes to generate transgenic strains. The methodology is well written in the overall sense, but lacks the clarity that correct genetic terminology can bring. In the detailed comments I provide, I have suggested corrections for the first time I encountered an issue. However, it they should be applied throughout the narrative and legends.

Impact statement:

"allow to create homozygous transgenic lines systematically" is awkward, may I suggest:

“allow us to systematically generate homozygous transgenic lines…”

Or

“allow the systematic construction of homozygous transgenic lines…”

Main text, first paragraph: “crossing setup[…]”; Do you mean, “crossing scheme”?

“allow to distinguish” is awkward, suggestion:

“allow one to distinguish”.

“purposely produced” is awkward, suggestion: “specifically designed”.

“phenotypically clearly distinguishable” – I understand the need for emphasis, but two adverbs seem like a little much.

Clearly distinguishable is much smoother and just as informative. After all aren't all transformation markers producing a phenotype?

"of both markers" a little clearer is "of the two markers".

"The progeny was" – throughout the paper you are using progeny in the plural (you could substitute children, not child). Thus it should be progeny were.

"which contains a mOrange-based6 and a mCherry-based7 eye-specific8 transformation marker". Suggestion: “That contains both mOrange-based and mCherry-based eye-specific transformation markers"

“appropriate excitation and emission”. Suggestion: “appropriate excitation and emission filters”.

"which were outcrossed against wild-type males" a little smoother and clearer "and these were crossed to".

Materials and methods:

“study utilizes a setup of vectors that are based on in silico designed and de novo synthetized sequences”. Suggestion: “study utilizes a set of vectors based on in silico design and de novo synthesis.”

“sublines were termed AGOC” – suggestion: “designated” or “called”

“Until this step, our route did not differ from most standard transgenic organisms establishment procedures.” A little smoother and clearer: “Through this step, our scheme did not differ from most standard procedures to generate transgenic organisms.”

"which carry mO and mC consecutively on the[…]" change to "that carry mO and mC in tandem".

"resulting in F4 mCe x mO-mC double hemizygotes in which Cre-mediated recombination occurs (Table 1, 'F3' row)" – this phrase actually follows from the previous sentence not the one to which it is attached. The genotype nomenclature is misleading, how about a completely new sentence:

“In the resulting F4 double hemizygotes (Ce; mO-mC), Cre-mediated recombination occurs (Table 1, 'F3' row).” Also please note the cross occurs in the F3, the F4 should be described (Ce; mO-mC) not mCe x mO-mC). Please use a semicolon to denote non-linkage.

Subsection “Systematic creation of homozygous lines”, same as above, the following is a cross not a genotype mCe x mO-mC. And "were outcrossed against wild-type males" Why are outcross and cross used interchangeably? It would be good to be strictly consistent in terminology usage.

Also, inbreeding is not the same as selfing. (Beetles cannot be selfed, since they are not hermaphrodites.) The naïve reader will appreciate the clarity if you check through entire manuscript for term usage consistency.

"F5 mO-only post-recombination hemizygous females were brother-sister crossed against F5 mC-only post recombination hemizygous males (Figure 2 and Figure 3, third row), resulting in F6 mO/mC heterozygotes, which carry once again both markers (Table 1, 'F5' row).” Since you are going to go into detail re cis/trans relationship of transgenes, why not use that terminology? May I suggest: “F5 mO-only post-recombination hemizygous females were mated to mC-only post recombination hemizygous male siblings (Figure 2 and Figure 3, third row), resulting in F6 mO/mC heterozygotes that carry both markers once again (Table 1, 'F5' row), but now in trans.”

"In contrast to the F3 mO-mC pre-recombination hemizygotes, which show the same phenotype but carry both markers consecutively on the maternal chromosome, the F6 mO/mC heterozygotes carry mO on the maternal and mC on the paternal chromosome. This was proven by crossing F6 mO/mC heterozygous females against wild-type males (Table 1, 'F6-S' row)." Suggestion: “In contrast to the F3 mO-mC pre-recombination hemizygotes, which display the same phenotype but carry both markers in tandem on the maternal chromosome, the F6 mO/mC heterozygotes carry mO on the maternal and mC on the paternal chromosome. This was demonstrated by crossing F6 mO/mC heterozygous females against wild-type males (Table 1, 'F6-S' row) and scoring the progeny." Please leave proofs to the mathematicians.

"F6 mO/mC heterozygous females were brother-sister crossed against genotypic identical F6 males (Figure 2 and Figure 3, fourth row), resulting in F7 mO- and mC-only homozygotes that carry either only mO or only mC on both, the maternal and paternal chromosomes (Table 1, 'F6' row).” This paragraph is very redundant and the figure references are a bit mixed up. May I suggest: "F6 mO/mC heterozygous females were mated with genotypically identical F6 male siblings (Figure 2 and Figure 3, fourth row). The resulting F7 homozygotes carry either only mO or only mC on both maternal and paternal chromosomes (Table 1, 'F6' row, and Figure 2 and Figure 3, fifth row), in contrast to the F5 mO- and mC-only post recombination hemizygotes, which display the same phenotype but carry either mO or mC on the maternal chromosome (Figure 2 and Figure 3, third row).”

Mated with or crossed to is better than crossed against.

“Proven” vs. “demonstrated” again.

"Although some phenotype distributions differed from the theoretical Mendelian values" – is the observed deviation significant?

"functionality" should be “function”

"inversed genders should be “reversed” or “opposite”.

Discussion: “exemplarily shown” should be “exemplified”.

“desynchronized” should be “unsynchronized”.

“it will perform” should be “it can be performed”…

A list of points should be just first, second, third, not firstly, secondly, and remove the word "alike".

"This works independently of" should be "independent of".

“we used the AGOC vector concept to systematically creating functional homozygous Tribolium lines that are designed for fluorescence live imaging of embryonic development.” – should be: “We used the AGOC vector concept to systematically create functional homozygous Tribolium lines[…]”

"Automation devices, equipped with a fluorescence detection unit, can be used to sort embryos according to their genotype". Although their phenotype does represent their hemizygous genotype, they are still sorted directly by their “phenotype" please correct this.

Materials and methods: "this study utilizes a setup of vectors that are based on in silico designed and de novo synthetized sequences." Suggestion: “this study utilizes a set of vectors based on in silico design and de novo synthethesis.”

“(ii) the four-slots (#1to #4) cloning site…." Should be “(ii) the four-slot (#1 to #4) cloning site” and change “evolved” to “derived”.

Change “scarless” to “scarlessly”.

Change “evolution” to “development” or “construction”.

Change “synthetized” to “synthesized”.

"and for all ligations the T4 DNA ligase (M0202L, New England BioLabs or provided with the pGEM-T Easy vector).” Change this to

“…and T4 DNA ligase (….) for all ligations.”

“One transformation vector was created that allowed the expression of a mEmerald-labeled sianyltransferase ubiquitously throughout the whole embryonic development.” This sentence (and all other similar sentences) is a little confusing. Suggestion: “We created one transformation vector that ubiquitously drives expression of mEmerald-labeled sianyltransferase throughout embryonic development.”

Did you name Plain-White As Snow? If so, you should take credit, if not please reference.

“(10 object slides with about 50 embryos reach, totaling at approximately 500 embryos per round[…])” Do you mean 50 embryos each? Also what is an object slide? Is that the same as a cover slip?

“All crossings were single-pair with one female and one male adult". Suggestion: “All crosses were performed with single male female pairs.”

“The progeny was singularized during the larval or pupal stages”. Suggestion: “Individual progeny were placed in new growth vials and scored for the presence…”

The last paragraph of the subsection “Crossing procedure, insert number determination cross and homozygous viability cross” seems redundant with the results and figures. Since this is a methods paper that is ok and the mating scheme does not need to be reiterated in the Materials and methods section. Only a description of the mating procedure (vials/media) needs to be described here.

"This strain derives from the white-eyed Pearl mutant strain39, but also carries the light ocular diaphragm mutation40, so that the adult eyes appear completely non pigmented". Suggestion: “For this study we generated a double mutant strain containing the Pearl and LOC mutations that result in completely unpigmented or white eyes, which we named PWAS.”

Figure 3 legend:

"The percentage boxes show the experimental (and theoretical) ratios of the progeny that show the respective phenotype". Suggestion: “The percentage boxes indicate the experimental (and theoretical) ratios of the progeny that display the respective phenotype.”

Figure 4 legend:

“their appearance change”, change to “their change in appearance”.

"Detail images show the" change to “Higher magnification images show…”

Table 1. It is difficult to distinguish the Bold vs. normal fonts. May I suggest a light background fill for those cells?

Supplemental notes:

"to a high stress levels" should "be high stress levels".

Therefore, those transgenic individuals are principally homozygous viable, but a certain degree might fail to develop properly, resulting in biased homozygous viability cross ratios". Do you mean the line is essentially viable, but a certain percent of individuals might fail to develop properly, producing biased ratios in the progeny of the homozygous viability cross? If so please correct.

Figure 3—figure supplement 2 legend: Change "one Cre line" to "a specific Cre line".

Figure 1—figure supplement 4: Change “Evolution” to “Derivation”.

"Vectors belong either to one or to two types, as indicated by the differently colored backgrounds." In the very next sentence you mention five types. What are the two types?

Supplementary Figure 11 legend: Change “used” to “documented”.

Supplementary file 10: change “proved” to “provided”.

Supplementary file 1: in the left column first cell "one insertions" should be "one insertion"

(and in all relevant tables). In the right column percentage works for the theoretical rows but does not need to be repeated in the rest of the cells of that column. That column actually enumerates the total number or (n) in each row.

"Segregation of mO-mC of 60% or less was defined as the criteria for one insert." A little smoother and clearer: “Segregation of 60% or fewer mO-mC beetles[…]”

"this theoretical value corresponds to the case that the insertions occur on different chromosomes, which is more likely than two independent inserts on the same chromosome" change to:”…to the case in which the insertions” or”…to the case where the insertions”,

Supplementary file 4:

1) “The background line of choice, i.e. PWAS in our study. After a few weeks of adulthood, a replacement F0 is grown from a separate egg collection procedure to keep the line alive.” Suggestion: “The background line of choice, i.e. PWAS in our study. After a few weeks of adulthood, a replacement F0 was grown from a separate oviposition to keep the line alive.”

2) “Approximately 1-2% of surviving embryos become F1 mosaics. They typically do not show any fluorescence and can only be identified by crossing them against the wild-type and analyzing the progeny.” Suggestion: “Approximately 1-2% of surviving embryos become F1 mosaics, which typically do not display any fluorescence and can only be identified by crossing to wild-type and analyzing the progeny.”

3) “Until this generation, our route does not differ from the standard transgenic animal establishment procedure. Working cultures can be established from the F3 individuals that can be treated like classical transgenic lines and used in (preliminary) experiments. Independently, the AGOC procedure is initiated by crossing F3 mO-mC pre-recombination hemizygous female against the mCe helper homozygote male which express the Cre recombinase.” Suggestion: “Through this generation, our scheme does not differ from the standard procedures to generate transgenic animals. Working cultures can be established from the F3 individuals that can be treated like classical transgenic lines and used in (preliminary) experiments. Alternatively, the AGOC procedure can be initiated by crossing an F3 mO-mC pre-recombination hemizygous female to an mCe helper homozygous male that expresses the Cre recombinase.”

4) "The F4 mCe×mO-mC double hemizygotes are hybrids. Within this generation, recombination occurs and one of both markers is excised. This process happens mainly during later stages of development, so that the individuals typically show a patchy transformation marker expression pattern, which is apparent in the adult eyes" Suggestion: “The F4 mC; mO-mC double hemizygotes are hybrids. Within this generation, recombination occurs and one of the two markers in cis is excised. This process usually happens during later stages of development, such that individuals typically display patchy transformation marker expression in adult eyes.”

5) “In the F5 generation, the Cre-expressing transgene as well as one of the transformation markers segregate away from the other transformation marker. As the transformation marker is typically excised after most of the germline cells have differentiated, F5 mO- and mC-only post-recombination hemizygotes are obtained from a single cross.” May I suggest: “In the F5 generation, the Cre-expressing transgene as well as one transformation marker are removed. F5 mO- and mC-only post-recombination hemizygotes are obtained from a single cross since the transformation marker is typically excised after most of the germline cells have differentiated.”

The second sentence describes what occurs to generate F5 progeny, so it actually happens in the F4, if you do not wish to move the sentence, it will be clearer to reverse the two phrases, so that it is clear when/where the excision event occurs.

Supplementary figure legends non-annotated should be unlabeled.

Supplementary file 12.

What is mounting agarose it is only mentioned in this table?

What is recipe for agarose platform?

Reviewer #3:

This work represents a nice implementation of site-specific recombination combined with fluorescent marker genes to initially allow identification of hemizygous transformants, and subsequently (after Cre-mediated recombination) allow recognition of homozygous or hemizygous individuals. The work is thorough and rigorous. The presentation of data is more than adequate to justify the conclusions. The authors demonstrate the utility of their method in Tribolium, but it should be applicable to almost any organism.

However, the concept presented by these authors is not exceptionally novel. For instance, "brainbow" methods involve much the same sort of recombination to generate mutually exclusive marker expression, although it is not usually applied to the germline.

Other significant concerns relate to the Discussion, both at the beginning, and at the end.

1) It seems to me that the best justification for this work is the ability to easily recognize homozygous mutants. However, the authors do not discuss this until the very end of the paper. The authors should mention this aspect at the beginning, even in the Abstract. Although there are other reasons to produce homozygotes (stock keeping, higher yield in experimental crosses, etc.), identifying homozygous mutants certainly has to be at the top of the list. The authors don't actually generate mutants in this paper, which would have been a nice addition, but it isn't critical to justify the utility of the technique.

2) In the Discussion, I don't fully accept all the justifications for the technique. For instance, their first reason, that it saves manpower because genotyping can be done by simple examination under a fluorescence microscope, seems a bit dubious. It is easy – yes, but look at how many generations of crossing it took to get to that point – seven. If homozygotes can be produced in four generations by standard techniques, how much work did it actually save?

3) In many cases, it may not be necessary to create homozygous lines. If transgenic animals are recognized by fluorescence, and all that is needed is an animal with the transgene, then hemizygotes may be good enough to carry out planned experiments. In fact, in some cases it may be better to use the hemizygous individual. For instance, random transgene insertion could generate a recessive mutation that, unknowingly, affects the phenotype under examination.

4) Can homozygotes be identified by fluorescence intensity at generation 4, instead of adding three more generations to cross to Cre-expressing animals, generate reduction events, and then identify homozygotes by intercrossing?

5) I really have no idea what is meant by "synchronized genotyping" or "desynchronized genotyping".

https://doi.org/10.7554/eLife.31677.041

Author response

Reviewer #1:

In this paper, Strobl et al. present a germline transformation protocol using a novel vector they've developed, that streamlines the process of making a transgene insert into the genome homozygous. The concept is simple yet novel and applicable, with modifications, to a wide range of animal genetic models that lack other means of easily homozygosing transgenes, such as by the use of balancers or targeted insertion sites. The authors demonstrate their technique using vectors they've constructed and fluorescent eye markers in the flour beetle, Tribolium. The technique works, and would appear to be a useful one for this beetle and other model genetic systems that lack advanced genetic tools. Overall the paper is well put together; it's complete and easy to understand and includes the tests needed to validate the method presented. However the paper's methods and figures sections are much longer and more detailed than they need to be, so I suggest simplifying these to make the whole more accessible to the interested reader. Specific comments follow below:

We are grateful for the positive comments. We agree with reviewer #1 and shortened the Materials and methods section (from ~3,300 to ~2.500 words) and reduced the number of supplementary display items (from 28 to 20).

Please see also comment 7.

1) Subsection “Systematic creation of homozygous lines”, first paragraph. To put things in perspective, this would take 3 generations in Drosophila using balancers, so a 4 generation procedure in Tribolium is not revolutionary, but rather an incremental advance.

We removed the term ‘only’ and added a comparison with the balancer-based approach to the Discussion.

For a detailed rationale, see reviewer #3 comment 2.

2) The complete DNA sequences of the vectors should be made available as supplemental data for this paper. This is quite important to make the method accessible to the scientific community.

We attached the complete and comprehensively annotated sequences for all 25 vectors as both, GeneBank (.gb) and Geneious (.geneious) files as Supplementary file 3 compressed into a single zipped folder (.zip). A reference is in the Supplementary Material section.

3) Would it not make sense to invert one of the marker genes in the vector to prevent read-through? Please comment.

We decided to have both markers on the same strand in tail-to-head orientation simply because this results in ‘real’, heterozygous individuals in the F6 generation, i.e. mO and mC are in the same orientation. Thereby, we avoid a read through between the marker and the mEmerald expression cassette of the functional transgenic lines in the F7 homozygotes after recombination.

4) In Figure 2, why is the first box F3 rather than F1 or F2? Please comment. Why is there a dotted line connecting rows 3 and 4, rather than solid?

Our paper reports a proof-on-principle study, thus the workflow was designed to be as logical/comprehensible as possible. With the F2 × wild-type cross, we demonstrate that the respective lines carry only a single insertion before starting with the mating procedure. Theoretically, the number of insertions can be determined by quantitatively evaluating a F2 × helper line cross, but since this is the very first study on this topic, we prefer to present an easily understandable workflow.

Following the same rationale, we need to keep a non-recombined culture of each transgenic line so that colleagues can request those lines and reproduce our results. If the F2 female is directly mated with the homozygous helper line, all descendants undergo recombination and we cannot establish a non-recombined culture.

For colleagues who utilize our vector concept, both above mentioned limitations do not apply. They may mate F2 founders directly with the Cre-expressing helper line. They can determine the insert number from the F2 × helper line cross, and they do not necessarily need a non-recombined culture of their respective transgenic lines. However, if a non-recombined culture is desired, we recommend to use a male instead of a female individual as founder in the F2 generation. The male can be used to inseminate a wild-type female for 2-3 days and can then be mated with a Cre-expressing helper female to start the procedure. We started the very first run with a female founder in the F3 generation and decided – for consistency – to start the mating procedure always with a female individual.

Once our concept is broadly used for more insect species as well as adapted to model organisms, we intend to develop a protocol that outlines an optimized procedure in which the current F3 generation is ‘skipped’.

Please see also comment 4 and reviewer #3 comment 2.

The dotted line is now explained within the figure legend.

5) Figures 3 and 4 are much more complex than necessary, and they didn't reproduce well. Please simplify these two figures.

We simplified Figure 3 by removing all images for the wild-type marker controls. The wild-type male in the F4, which is mated with the double hemizygous female, now also functions as the marker control (which is also indicated).

We think that the creation of homozygous transgenic lines for fluorescence live imaging assays is an important field of application for our concept, and that we also have to prove the functionality of the respective mEmerald-labeled Lifeact vectors beyond any doubt by providing a glimpse at our live imaging data. However, we understand that this data should not distract from the actual topic of our study, thus we strongly reduced the size of Figure 4. We are convinced that the figure is now at the practicable minimum – we still want to show that our vectors/transgenic lines perform adequately in long-term live imaging assays.

We are also convinced that the performance of novel transgenic lines in live imaging assays is of importance for the Tribolium community. In our study, we present the very first filamentous actin-labeled transgenic Tribolium lines and provide valuable information on two previously uncharacterized promoter sequences, Zen1 and ARP5.

6) The text in Table 1 is too small to read. Could this be presented in a more intuitive form?

We are well aware that Table 1 is relatively large, but it is the centerpiece of the study. We already designed the table as concisely as possible, and any restructuring would meddle with the distinctness. The final layout lies within the discretion of eLife. We will inquire if it is possible to print this table in landscape format, alternatively the small images that represent the genotypes can be removed.

7) The supplementary data are longer and more complex than necessary. Please include only what is necessary to convey the method to the reader.

We significantly reduced the number of supplementary display items. We simplified (similarly to Figure 3) and/or combined Supplementary Figures 3 and 4 [now: Figure 3—figure supplement 2] and removed Supplementary Figures 6 and 11. We also simplified and combined Supplementary Tables 5 and 6, 1 and 9, 2 and 10, 3 and 11 as well as 13 and 16. In total, 8 supplementary display items were removed while the internal logic and overall consistency were improved.

8) In the Discussion, please include a list of the organisms in which this technique might be useful, and discuss how the vector would have to be modified to be used in these other organisms. Discussing these issues is important to ensure adoption of this method by the broader research community.

Reviewer #1 raises an important concern here. We already briefly discussed adaption to other model organisms, but we agree that our argumentation can be expanded. In the revised manuscript, we discuss adaption to other insect model organisms, zebrafish and mouse in more detail.

Reviewer #2:

The authors describe important technical improvements to transgenic vector constructs that allow efficient production of homozygous transgenic strains. Their concept is applicable to any system in which one wishes to generate transgenic strains. The methodology is well written in the overall sense, but lacks the clarity that correct genetic terminology can bring. In the detailed comments I provide, I have suggested corrections for the first time I encountered an issue. However, it they should be applied throughout the narrative and legends.

We appreciate the very positive evaluation of our vector concept. Furthermore, we would like to express our gratitude for the comprehensive valuable suggestions on how to improve the nomenclature and scientific conciseness.

Impact statement:

"allow to create homozygous transgenic lines systematically" is awkward, may I suggest:

“allow us to systematically generate homozygous transgenic lines[…]”

Or

“allow the systematic construction of homozygous transgenic lines[…]”

We changed the impact statement accordingly.

Main text, first paragraph: “crossing setup[…]” Do you mean “crossing scheme”?

We changed the term ‘setup’ to ‘scheme’.

“allow to distinguish” is awkward, suggestion:

“allow one to distinguish”.

“purposely produced” is awkward, suggestion: “specifically designed”.

Both incongruities were corrected, the first sentence was rephrased to improve the overall conciseness, the second one was changed as suggested.

“phenotypically clearly distinguishable” – I understand the need for emphasis, but two adverbs seem like a little much.

Clearly distinguishable is much smoother and just as informative. After all aren't all transformation markers producing a phenotype?

We agree with the argumentation of reviewer #2 here and removed ‘phenotypically’ from this sentence and from the Abstract.

"of both markers" a little clearer is "of the two markers".

"The progeny was" – throughout the paper you are using progeny in the plural (you could substitute children, not child). Thus it should be progeny were.

We corrected respective occurrences throughout the whole manuscript.

"which contains a mOrange-based6 and a mCherry-based7 eye-specific8 transformation marker". Suggestion: “That contains both mOrange-based and mCherry-based eye-specific transformation markers".

The sentence was corrected accordingly.

“appropriate excitation and emission”. Suggestion: “appropriate excitation and emission filters”.

We rephrased the sentence as follows: ‘Both fluorescent proteins are spectrally separable by appropriate excitation bands and emission filters.’

"which were outcrossed against wild-type males" a little smoother and clearer "and these were crossed to".

We removed the term ‘outcrosses against’ completely throughout the manuscript and constantly use the term ‘mated with’ now.

Materials and methods:

“study utilizes a setup of vectors that are based on in silico designed and de novo synthetized sequences”. Suggestion: “study utilizes a set of vectors based on in silico design and de novo synthesis.”

The sentence was shortened according to the suggestion.

“sublines were termed AGOC” – suggestion: “designated” or “called”.

We replaced ‘termed’ with ‘called’ in all occurrences except for the designation of vectors and genetic elements.

“Until this step, our route did not differ from most standard transgenic organisms establishment procedures.” A little smoother and clearer: “Through this step, our scheme did not differ from most standard procedures to generate transgenic organisms.”

We changed the sentence to ‘Up to this step, our scheme did not differ from most standard transgenic line establishment procedures.’

"which carry mO and mC consecutively on the[…]" change to "that carry mO and mC in tandem".

We changed the sentence to ‘…that carry mO and mC in cis configuration…’

"resulting in F4 mCe x mO-mC double hemizygotes in which Cre-mediated recombination occurs (Table 1, 'F3' row)" – this phrase actually follows from the previous sentence not the one to which it is attached. The genotype nomenclature is misleading, how about a completely new sentence:

“In the resulting F4 double hemizygotes (Ce; mO-mC), Cre-mediated recombination occurs (Table 1, 'F3' row).” Also please note the cross occurs in the F3, the F4 should be described (Ce; mO-mC) not mCe x mO-mC). Please use a semicolon to denote non-linkage.

We rephrased this paragraph as well as the previous paragraph slightly. Information about the helper line is now provided in the previous paragraph.

Subsection “Systematic creation of homozygous lines”, same as above, the following is a cross not a genotype mCe x mO-mC. And "were outcrossed against wild-type males" Why are outcross and cross used interchangeably? It would be good to be strictly consistent in terminology usage.

Also, inbreeding is not the same as selfing. (Beetles cannot be selfed, since they are not hermaphrodites.) The naïve reader will appreciate the clarity if you check through entire manuscript for term usage consistency.

We removed the term ‘outcross’ completely.

"F5 mO-only post-recombination hemizygous females were brother-sister crossed against F5 mC-only post recombination hemizygous males (Figure 2 and Figure 3, third row), resulting in F6 mO/mC heterozygotes, which carry once again both markers (Table 1, 'F5' row).” Since you are going to go into detail re cis/trans relationship of transgenes, why not use that terminology? May I suggest: “F5 mO-only post-recombination hemizygous females were mated to mC-only post recombination hemizygous male siblings (Figure 2 and Figure 3, third row), resulting in F6 mO/mC heterozygotes that carry both markers once again (Table 1, 'F5' row), but now in trans.”

We changed the sentence, but prefer to use the term ‘trans configuration’. We also appreciate the suggestion to use the term ‘siblings’ to ‘instead of the term ‘brother-sister’ to simplify this and similar sentences throughout our manuscript. We still prefer to keep the first sentence short and explain the marker configuration in a second sentence.

"In contrast to the F3 mO-mC pre-recombination hemizygotes, which show the same phenotype but carry both markers consecutively on the maternal chromosome, the F6 mO/mC heterozygotes carry mO on the maternal and mC on the paternal chromosome. This was proven by crossing F6 mO/mC heterozygous females against wild-type males (Table 1, 'F6-S' row)." Suggestion: “In contrast to the F3 mO-mC pre-recombination hemizygotes, which display the same phenotype but carry both markers in tandem on the maternal chromosome, the F6 mO/mC heterozygotes carry mO on the maternal and mC on the paternal chromosome. This was demonstrated by crossing F6 mO/mC heterozygous females against wild-type males (Table 1, 'F6-S' row) and scoring the progeny." Please leave proofs to the mathematicians.

We removed the first sentence (‘In contrast to[…]’) from the manuscript to avoid confusion. We changed the second sentence accordingly and used the terms ‘display’, ‘demonstrate’ and ‘scoring’ in other sections of our manuscript.

"F6 mO/mC heterozygous females were brother-sister crossed against genotypic identical F6 males (Figure 2 and Figure 3, fourth row), resulting in F7 mO- and mC-only homozygotes that carry either only mO or only mC on both, the maternal and paternal chromosomes (Table 1, 'F6' row).” This paragraph is very redundant and the figure references are a bit mixed up. May I suggest: "F6 mO/mC heterozygous females were mated with genotypically identical F6 male siblings (Figure 2 and Figure 3, fourth row). The resulting F7 homozygotes carry either only mO or only mC on both maternal and paternal chromosomes (Table 1, 'F6' row, and Figure 2 and Figure 3, fifth row), in contrast to the F5 mO- and mC-only post recombination hemizygotes, which display the same phenotype but carry either mO or mC on the maternal chromosome (Figure 2 and Figure 3, third row).”

Mated with or crossed to is better than crossed against.

We simplified the paragraph by removing the ‘In contrast to[…]’ sentence to avoid confusion and changed the remaining sentences according to the suggestions.

For consistency, we stick to the term ‘mated with’.

“Proven” vs. “demonstrated” again.

We removed the term ‘prove’ nearly completely from the manuscript. We only kept the term ‘proof-of-principle’.

"Although some phenotype distributions differed from the theoretical Mendelian values" – is the observed deviation significant?

The differences are not significant. We rephrased the sentence to make our point clearer: “Throughout all generations, the subline-specific scores matched the expectations and no significant differences between the respective arithmetic means and the theoretical Mendelian ratios were found. Importantly, all expected phenotypes, and thus all expected genotypes, were found in all generations and F7 (mO/mO) as well as (mC/mC) homozygotes were obtained for all six AGOC sublines.”

We also added a sentence to the Materials and methods section that explains the statistical test that we performed, a notification to the description of Table 1 and the r respective supplementary tables. For convenience, the standard deviations were also added to the arithmetic mean rows in Table 1 and the respective supplementary tables.

"functionality" should be “function”

We corrected the issue

"inversed genders should be “reversed” or “opposite”.

Discussion: “exemplarily shown” should be “exemplified”.

“desynchronized” should be “unsynchronized”.

“it will perform” should be “it can be performed”.

A list of points should be just first, second, third, not firstly, secondly, and remove the word "alike".

"This works independently of" should be "independent of".

All of those minor issues were resolved. We agree with reviewer #2 on the first concern, but changed ‘inversed genders’ to ‘swapped genders’ since we would like to avoid using genetic-related (e.g. ‘inverted terminal repeats’, ‘reverse transcription’) nomenclature here.

We also replaced Firstly etc. by Roman numerals within brackets.

Please see also reviewer #3 comment 5.

“we used the AGOC vector concept to systematically creating functional homozygous Tribolium lines that are designed for fluorescence live imaging of embryonic development.” – should be: “We used the AGOC vector concept to systematically create functional homozygous Tribolium lines[…]”

The mistake was corrected.

"Automation devices, equipped with a fluorescence detection unit, can be used to sort embryos according to their genotype". Although their phenotype does represent their hemizygous genotype, they are still sorted directly by their “phenotype" please correct this.

We are thankful for this important remark and changed the sentence to: ‘Automation devices, equipped with a fluorescence phenotype-adapted detection unit, in our case fluorescence, can be used to sort embryos organisms with different genotypes according to their markers’.

Materials and methods: "this study utilizes a setup of vectors that are based on in silico designed and de novo synthetized sequences." Suggestion: “this study utilizes a set of vectors based on in silico design and de novo synthethesis.”

We agree that the suggested shorter sentence also comes with an increase in legibility.

“(ii) the four-slots (#1to #4) cloning site[…]." Should be “(ii) the four-slot (#1 to #4) cloning site”.

We corrected the issue.

And change “evolved” to “derived”.

Change “scarless” to “scarlessly”.

Change “evolution” to “development” or “construction”.

Change “synthetized” to “synthesized”.

All of those minor issues were corrected either by removing the respective sentences as part of the Materials and methods shortening procedure or by correcting the issue.

"and for all ligations the T4 DNA ligase (M0202L, New England BioLabs or provided with the pGEM-T Easy vector).” Change this to

“[…]and T4 DNA ligase (….) for all ligations.”

We accept this suggestion and also changed the sentence accordingly.

“One transformation vector was created that allowed the expression of a mEmerald-labeled sianyltransferase ubiquitously throughout the whole embryonic development.” This sentence (and all other similar sentences) is a little confusing. Suggestion: “We created one transformation vector that ubiquitously drives expression of mEmerald-labeled sianyltransferase throughout embryonic development.”

We deleted this sentence (and other, similar sentences) as part of the Materials and methods shortening procedure.

Did you name Plain-White As Snow? If so, you should take credit, if not please reference.

We rephrased the sentence to make our point clearer.

“(10 object slides with about 50 embryos reach, totaling at approximately 500 embryos per round[…])” Do you mean 50 embryos each? Also what is an object slide? Is that the same as a cover slip?

We rephrased the sentence to make our point clearer.

“All crossings were single-pair with one female and one male adult". Suggestion: “All crosses were performed with single male female pairs.”

We adopted the suggestion.

“The progeny was singularized during the larval or pupal stages”. Suggestion: “Individual progeny were placed in new growth vials and scored for the presence[…]”

We also adopt this suggestion, but slightly rephrased the sentence.

The last paragraph of the subsection “Crossing procedure, insert number determination cross and homozygous viability cross” seems redundant with the results and figures. Since this is a methods paper that is ok and the mating scheme does not need to be reiterated in the Materials and methods section. Only a description of the mating procedure (vials/media) needs to be described here.

We followed the recommendation of reviewer #2 and replaced several sentences with a brief statement that the information is found within the Results section.

"This strain derives from the white-eyed Pearl mutant strain39, but also carries the light ocular diaphragm mutation40, so that the adult eyes appear completely non pigmented". Suggestion: “For this study we generated a double mutant strain containing the Pearl and LOC mutations that result in completely unpigmented or white eyes, which we named PWAS.”

We approve the rephrasing suggestion, but prefer to not abbreviate light ocular diaphragm.

Figure 3 legend:

"The percentage boxes show the experimental (and theoretical) ratios of the progeny that show the respective phenotype". Suggestion: “The percentage boxes indicate the experimental (and theoretical) ratios of the progeny that display the respective phenotype.”

We accept the suggestion and adapt it for similar occasions.

Figure 4 legend:

“their appearance change”, change to “their change in appearance”.

"Detail images show the" change to “Higher magnification images show[…]”

We changed ‘detail’ to ‘enlarged’. We prefer not to use the term ‘magnification’ here, since this might be misunderstood as ‘we switched to a higher magnification objective’.

Table 1. It is difficult to distinguish the Bold vs. normal fonts. May I suggest a light background fill for those cells?

We agree with reviewer #2 here, but we do not know of this will be in compliance with the eLife house style, since most of the tables that we saw within eLife articles only have shading in the header in the online version and no shading at all in the PDF version. If it is not possible, we would prefer to stick to bold font.

Supplemental notes:

"to a high stress levels" should "be high stress levels".

We corrected the typo.

Therefore, those transgenic individuals are principally homozygous viable, but a certain degree might fail to develop properly, resulting in biased homozygous viability cross ratios". Do you mean the line is essentially viable, but a certain percent of individuals might fail to develop properly, producing biased ratios in the progeny of the homozygous viability cross? If so please correct.

Yes. We rephrased the sentence.

Figure 3—figure supplement 2 legend: Change "one Cre line" to "a specific Cre line".

Figure 1—figure supplement 4: Change “Evolution” to “Derivation”.

Both corrections were made.

"Vectors belong either to one or to two types, as indicated by the differently colored backgrounds." In the very next sentence you mention five types. What are the two types?

We changed the sentences slightly to clarify that ‘individual vectors belong to either one or two of five different types’.

Supplementary Figure 11 legend: Change “used” to “documented”.

Supplementary file 10: change “proved” to “provided”.

Supplementary Figure 11 was removed, so a change is no longer necessary.

The second remark was corrected.

Supplementary file 1: in the left column first cell "one insertions" should be "one insertion" (and in all relevant tables). In the right column percentage works for the theoretical rows but does not need to be repeated in the rest of the cells of that column. That column actually enumerates the total number or (n) in each row.

We corrected the typo and removed the ‘100%’ entries from the table and all other tables.

"Segregation of mO-mC of 60% or less was defined as the criteria for one insert." A little smoother and clearer: “Segregation of 60% or fewer mO-mC beetles[…]”

We changed the wording in this and the following supplementary table.

"this theoretical value corresponds to the case that the insertions occur on different chromosomes, which is more likely than two independent inserts on the same chromosome" change to: “[…]to the case in which the insertions” or”[…]to the case where the insertions”.

We changed the sentence accordingly.

Supplementary file 4:

1) “The background line of choice, i.e. PWAS in our study. After a few weeks of adulthood, a replacement F0 is grown from a separate egg collection procedure to keep the line alive.” Suggestion: “The background line of choice, i.e. PWAS in our study. After a few weeks of adulthood, a replacement F0 was grown from a separate oviposition to keep the line alive.”

We accept the suggestion.

2) “Approximately 1-2% of surviving embryos become F1 mosaics. They typically do not show any fluorescence and can only be identified by crossing them against the wild-type and analyzing the progeny.” Suggestion: “Approximately 1-2% of surviving embryos become F1 mosaics, which typically do not display any fluorescence and can only be identified by crossing to wild-type and analyzing the progeny.”

We rephrased the sentence according to the suggestion and improved the overall description.

3) “Until this generation, our route does not differ from the standard transgenic animal establishment procedure. Working cultures can be established from the F3 individuals that can be treated like classical transgenic lines and used in (preliminary) experiments. Independently, the AGOC procedure is initiated by crossing F3 mO-mC pre-recombination hemizygous female against the mCe helper homozygote male which express the Cre recombinase.” Suggestion: “Through this generation, our scheme does not differ from the standard procedures to generate transgenic animals. Working cultures can be established from the F3 individuals that can be treated like classical transgenic lines and used in (preliminary) experiments. Alternatively, the AGOC procedure can be initiated by crossing an F3 mO-mC pre-recombination hemizygous female to an mCe helper homozygous male that expresses the Cre recombinase.”

We accept the suggestion, but modified the sentence slightly.

4) "The F4 mCe×mO-mC double hemizygotes are hybrids. Within this generation, recombination occurs and one of both markers is excised. This process happens mainly during later stages of development, so that the individuals typically show a patchy transformation marker expression pattern, which is apparent in the adult eyes" Suggestion: “The F4 mC; mO-mC double hemizygotes are hybrids. Within this generation, recombination occurs and one of the two markers in cis is excised. This process usually happens during later stages of development, such that individuals typically display patchy transformation marker expression in adult eyes.”

We approve the suggestion, rephrased the sentence, but stick to the term ‘cis configuration’.

5) “In the F5 generation, the Cre-expressing transgene as well as one of the transformation markers segregate away from the other transformation marker. As the transformation marker is typically excised after most of the germline cells have differentiated, F5 mO- and mC-only post-recombination hemizygotes are obtained from a single cross.” May I suggest: “In the F5 generation, the Cre-expressing transgene as well as one transformation marker are removed. F5 mO- and mC-only post-recombination hemizygotes are obtained from a single cross since the transformation marker is typically excised after most of the germline cells have differentiated.”

The second sentence describes what occurs to generate F5 progeny, so it actually happens in the F4, if you do not wish to move the sentence, it will be clearer to reverse the two phrases, so that it is clear when/where the excision event occurs.

Supplementary figure legends non-annotated should be unlabeled.

We exchanged the terms as suggested.

Supplementary file 12.

What is mounting agarose it is only mentioned in this table?

All developmental biology-associated live imaging assays with light sheet-based fluorescence microscopy use mounting agarose to form a stable mounting matrix (a column, a block, a cup, a hemisphere, a thin film, a pocket…). Protocols for insect embryos are actually well-established (Drosophila: Keller et al. 2011, Cold Spring Harbor Protocols; Schmied and Tomancak 2016, Methods in Molecular Biology), and two protocols for Tribolium are cited within the Materials and methods section. We improved the table by adding another row that references the detailed mounting technique.

What is recipe for agarose platform?

We made slight adjustments to the sentence to clarify our point.

Reviewer #3:

This work represents a nice implementation of site-specific recombination combined with fluorescent marker genes to initially allow identification of hemizygous transformants, and subsequently (after Cre-mediated recombination) allow recognition of homozygous or hemizygous individuals. The work is thorough and rigorous. The presentation of data is more than adequate to justify the conclusions. The authors demonstrate the utility of their method in Tribolium, but it should be applicable to almost any organism.

However, the concept presented by these authors is not exceptionally novel. For instance, "brainbow" methods involve much the same sort of recombination to generate mutually exclusive marker expression, although it is not usually applied to the germline.

We only partially agree with reviewer #3 here. The functions of both concepts are entirely different and not comparable. Brainbow was developed to aid on the ‘cellular level’. It has nothing to do with genotyping, husbandry and the systematic creation of homozygous transgenic lines but supports the identification and characterization process of individual neurons and their respective dendrites and axons in a single organism. Our concept was developed to aid on the ‘organismal level’. It has nothing to do with differences amongst individual cells but facilitates the handling of the many multi-cellular individuals that are required to maintain the line and obtain descendants with the desired genotype.

Other significant concerns relate to the Discussion, both at the beginning, and at the end.

1) It seems to me that the best justification for this work is the ability to easily recognize homozygous mutants. However, the authors do not discuss this until the very end of the paper. The authors should mention this aspect at the beginning, even in the Abstract. Although there are other reasons to produce homozygotes (stock keeping, higher yield in experimental crosses, etc.), identifying homozygous mutants certainly has to be at the top of the list. The authors don't actually generate mutants in this paper, which would have been a nice addition, but it isn't critical to justify the utility of the technique.

We strongly agree with reviewer #3 that workflows, which involve the creation of homozygous mutants, independent the actual method – e.g. large scale insertional mutagenesis (Trauner et al. 2009, BMC Biology) or genome engineering (Gilles et al., 2015) – can benefit from our vector concept, which does not only apply to Tribolium but also to other model organisms. A study in which the AGOC vector concept is used as part of a mutagenesis / knock-out assay can be expected in the near future. We added a short sentence to the Abstract.

2) In the Discussion, I don't fully accept all the justifications for the technique. For instance, their first reason, that it saves manpower because genotyping can be done by simple examination under a fluorescence microscope, seems a bit dubious. It is easy – yes, but look at how many generations of crossing it took to get to that point – seven. If homozygotes can be produced in four generations by standard techniques, how much work did it actually save?

We agree with reviewer #3 that in certain scenarios, for some aspects, our concept is rivaled by other techniques, ‘waiting time’ being the obvious one. We are well aware of this, thus we intentionally avoid the term ‘time’ and rather justify our approach by discussing manpower (i.e. the total ‘working time’ the scientist is busy performing the respective assays) and consumables. Since genotyping and homozygosing is an important, yet very complex and extremely heterogeneous topic (which model organism, which transgenesis approach and which genotyping / homozygosing strategy is used?), a comprehensive comparison lies beyond the scope of the discussion. Our concept complements and improves the arsenal of genotyping and homozygosing techniques. We put a lot of effort into explaining the architecture and functionality as detailed as possible so that interested readers can decide whether our suggestion benefits their workflow or whether another approach should be chosen.

Since eLife also publishes the decision letter and the author response, we would like to provide a few more thoughts that may be of relevance for interested colleagues:

- In Drosophila, when site-specific integration is used, balancer-based homozygosing requires less ‘waiting time’ in comparison to our concept. However, in Tribolium and mouse, only very few, and for zebrafish, basically no balancers are available. Additionally, usage of balancers increases recombination in non-balancer regions, the respective balancer lines have to be maintained in the laboratory (or obtained from a stock center or colleague) and certain balancers may also not be available for the background strain of choice. Our concept was established with universality in mind and thus has to be judged from multiple points of view and by considering all relevant circumstances.

- When random integration is performed, the few available balancers are only a convenient choice when the insertion site is known. Genotyping via genetic assays also requires data on the insertion site, leaving test crossing assays as the only option. Test crossing by itself also has severe drawbacks.

- When a large number of newly established transgenic lines have to be handled, ‘waiting time’ is usually only a minor issue, while manpower as well as resources become major challenges and thus also the scope-limiting factors. Recently, we also established a genetic assay-based non-lethal genotyping protocol for Tribolium (Strobl et al., 2017) and are thus able to compare both approaches at first hand, confirming that the savings in manpower and resources are significant.

- This is a proof-of-principle study, therefore we decided to demonstrate the functionality of our concept as ‘clean’ as possible for 7 generations. At least three ‘quick and dirty shortcuts’ can be considered:

i) The F3 generation is optional, F2 founders can be directly mated with the Cre-expressing helper lines. Since we have to keep a non-recombination culture of all transgenic lines for reasons of reproduction, we had to add another generation.

ii) F4 [now F3] (mCe; mO-mC) double hemizygotes can be mated with genotypically identical siblings, resulting in 6.25% F6 [now F4] (mO/mC) heterozygotes.

iii) F6 [now F4] (mO/mC) heterozygotes are, in basically all experimental scenarios, functionally identical with F7 (mO/mO) homozygotes / F7 (mC/mC) homozygotes.

- Workflow complexity should also not be underestimated. After a brief introduction, the procedure that we outline within our manuscript can be robustly conducted by student assistants.

See also reviewer #1 comments 1 and 4.

We removed one occurrence of the term ‘time’ and rephrased the respective sentence. We also mention the balancer-based approach briefly within the Discussion.

3) In many cases, it may not be necessary to create homozygous lines. If transgenic animals are recognized by fluorescence, and all that is needed is an animal with the transgene, then hemizygotes may be good enough to carry out planned experiments. In fact, in some cases it may be better to use the hemizygous individual. For instance, random transgene insertion could generate a recessive mutation that, unknowingly, affects the phenotype under examination.

We only partially agree with reviewer #3 here. In theory, there are assays in which hemizygotes are the preferred choice and other assays in which homozygotes are the preferred choice. However, e.g. in fluorescence live imaging assays using transgenic lines, there are typically two practical cases: either the embryo collection culture is homozygous, and thus all progeny is homozygous, or the culture is mixed (hemi- and homozygotes, eventually also wild-types), and the genotype of the descendants may remain unknown. This is absolutely not recommended since it adds another uncontrollable parameter to the assay which might have a substantial influence on the results.

The concern that reviewer #3 raises here is actually one of the major advantages of our concept. The experimenter can systematically evaluate if the transgene insertion into a random location leads to a phenotype when homozygously present. Thus, the effect becomes known, and the experimenter can continue to work with hemizygous individuals or create more transgenic sublines in which the transgene does not provoke a phenotype when homozygously present.

4) Can homozygotes be identified by fluorescence intensity at generation 4, instead of adding three more generations to cross to Cre-expressing animals, generate reduction events, and then identify homozygotes by intercrossing?

In Tribolium (and probably also all in other model organisms) this would not reliably work. For most of the transgenic lines, the mean expression levels of the markers do not obviously differ between hemizygous and homozygous individuals. For a few other lines, the mean expression levels of homozygotes appear to be slightly higher than for hemizygotes. However, due to individual plasticity and most probably other factors such as age and maybe also gender, homozygotes and hemizygotes have broad signal strength windows with a large overlap. Thus, some (weakly expressing) homozygotes show less signal than some (strongly expressing) hemizygotes. Also, such an approach would not work very well in conjunction with automation.

In biology, it is always problematic to reliably distinguish between ‘some’ and ‘some more’, especially when both windows have a large overlap. With our concept, the experimenter has to differentiate between ‘none’ and ‘some’, which do not overlap at all. This is the most convenient and most reliable approach.

5) I really have no idea what is meant by "synchronized genotyping" or "desynchronized genotyping".

We changed the term ‘desynchronized’ to ‘unsynchronized’ and improved the sentences by clarifying that we refer to multiple individuals.

https://doi.org/10.7554/eLife.31677.042

Article and author information

Author details

  1. Frederic Strobl

    Physical Biology, BMLS, CEF-MC, Goethe Universität, Frankfurt am Main, Germany
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    frederic.strobl@physikalischebiologie.de
    Competing interests
    Patent application for the vector concept (DE file number: 10 2017 112 863.8).
  2. Anita Anderl

    Physical Biology, BMLS, CEF-MC, Goethe Universität, Frankfurt am Main, Germany
    Contribution
    Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Ernst HK Stelzer

    Physical Biology, BMLS, CEF-MC, Goethe Universität, Frankfurt am Main, Germany
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    ernst.stelzer@physikalischebiologie.de
    Competing interests
    Ernst HK Stelzer: Patent application for the vector concept (DE file number: 10 2017 112 863.8).
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1545-0736

Funding

Deutsche Forschungsgemeinschaft (CEF-MC, EXC 115)

  • Frederic Strobl
  • Anita Anderl
  • Ernst HK Stelzer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank J Alexander Ross, Katharina Hötte, Berit Reinhardt and Sigrun Becker for their valuable support. The pBSII-IFP-ORF vector was a kind gift from Malcom Fraser (University of Notre Dame, Indiana, United States).

Reviewing Editor

  1. Bruce Edgar, University of Utah, United States

Publication history

  1. Received: August 31, 2017
  2. Accepted: January 29, 2018
  3. Version of Record published: March 15, 2018 (version 1)
  4. Version of Record updated: March 20, 2018 (version 2)

Copyright

© 2018, Strobl et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,985
    Page views
  • 215
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Developmental Biology
    2. Physics of Living Systems
    Silas Boye Nissen et al.
    Research Article Updated
    1. Developmental Biology
    Debadrita Bhattacharya et al.
    Research Article