One of the most powerful techniques for characterizing gene function is to generate transgenic animals in which an epitope tag such as GFP has been fused to the gene at its normal genomic location (Ross-Macdonald et al., 1999; Morin et al., 2001; Skarnes et al., 2004). These tagged proteins are extremely useful as they permit determination of protein localization in vivo as well as conditional, tissue specific, temporal and reversible removal of the tagged proteins (Nagarkar-Jaiswal et al., 2015). However, previous methods for generating protein trap alleles in Drosophila have allowed only about 800 genes to be successfully tagged (Kelso et al., 2004; Buszczak et al., 2007; Quinones-Coello et al., 2007; Aleksic et al., 2009; Lowe et al., 2014).

We previously developed a flexible system for engineering the Drosophila genome using the Minos-Mediated Integration Cassette (MiMIC) transposable element. We generated 15,660 strains with a single MiMIC inserted at random within the fly genome and mapped their insertion site (Bellen et al., 2011; Venken et al., 2011; Nagarkar-Jaiswal et al., 2015). MiMIC carries sequences that function as a gene and protein trap when inserted in the proper orientation in a coding intron. Moreover, its content can be replaced by Recombination-Mediated Cassette Exchange (RMCE) leading to the introduction of any desired DNA, such as an artificial exon that encodes a protein tag. This approach can potentially be used to tag thousands of genes. Currently, 2854 existing insertions are located within the coding introns of 1862 distinct genes (Nagarkar-Jaiswal et al., 2015), and MiMIC-like elements can now be placed in any gene of interest by CRISPR (Zhang et al., 2014). Unfortunately, the RMCE method needed to convert these insertions into functional protein traps requires embryonic injections of an appropriate donor DNA and screening of many offspring to identify the desired events, a labor and cost-intensive procedure that does not scale easily. We therefore developed a more efficient and economical in vivo genetic tagging methodology that can in principle be used to generate protein trap alleles of all Drosophila genes.

We developed a genetic strategy that allows the desired RMCE event to take place efficiently without the need for microinjection. The method uses FLP recombinase to release a genomically integrated DNA flanked by FRT sites into the nucleoplasm where it can efficiently undergo phiC31 integrase-mediated cassette exchange, as shown by Gohl et al. (2011). As shown in Figure 1A, we engineered three donor cassettes, one for each reading frame. The core, which contains a splice acceptor (SA) followed by a (GGS)₄ flexible linker, multiple tags (EGFP-FlAsH-StrepII-TEVcs-3xFlag {GFSTF}), another (GGS)₄ flexible linker, and a splice donor (SD), is flanked by two inverted attB sites for phiC31-mediated RMCE (Venken et al., 2011). We then cloned this cassette core between tandem FRT sites in a P-element transformation vector (Gong and Golic, 2003). FLP-mediated recombination between the tandem FRT sites excises a circular donor DNA molecule from its initial genetic locus, promoting its efficient recombination with a distal target site (Golic et al., 1997). A mini-white eye color marker gene between our donor cassette and one of the FRT sites allows us to monitor the presence or absence of the donor cassette in FLP recombinase-containing stocks.

Figure 1

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Figure 1—source data 1

List of constructs.

https://doi.org/10.7554/eLife.08469.003

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Figure 1—source data 2

List of fly strains generated.

Stocks #1 to 6 are stocks that contain the GFSTF construct (phase 0, 1, and 2) for tagging genes/proteins that map to chromosome II and III. Stocks # 7: FRT cassette maps to chromosome 2 and contains phase 0 construct; #8: FRT cassette maps to chromosome 2 and contains phase 1 construct; #9: FRT cassette maps to chromosome 2 and contains phase 2 construct; #10: FRT cassette maps to chromosome 3 and contains phase 0 construct; #11: FRT cassette maps to chromosome 3 and contains phase 1 construct; #12 FRT cassette maps to chromosome 3 and contains phase 2 construct.

https://doi.org/10.7554/eLife.08469.004

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We created 6 stocks (Figure 1—source data 2), each harboring one of the three donor transgenes located on the second or third chromosome, and a heat shock-inducible FLP recombinase and a germ line-expressed phiC31 integrase on the X-chromosome. Because the heat shock-inducible FLP recombinase is somewhat leaky at 18°C, the donor transgene is lost from these stocks at a low frequency, resulting in rare white-eyed flies, which we periodically discard.

To initiate RMCE, we crossed the appropriate donor flies to MiMIC-containing flies and heat shocked the resulting embryos and larvae (Figure 2). Within the primordial germ cells of some of the embryos and larvae, phiC31 integrase catalyzed recombination between attB sites in the donor and attP sites in the MiMIC transposon. The positive RMCE events were selected based on the loss of the y + marker present in the original MiMIC (Figure 2). We confirmed the integration and orientation of the donor cassette by PCR as described in Venken et al. (2011). Typically, 50% of the integration events are in the proper orientation.

Figure 2

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We observed one to ten RMCE events in 93 out of 113 attempts in our initial trial when we set up 3–7 crosses (Cross 2 in Figure 2). After PCR screening, 60/93 of the tested MiMICs allowed integration of at least one donor in the proper orientation to tag the endogenous gene (Supplementary file 1). In summary, we set up 3–7 vials for each starting cross and obtained 60/113 tagged genes. Since the efficiency of RMCE and the ease of detecting yellow⁻ progeny vary between different starting sites, we propose to set up 10–20 vials and to score more progeny to improve the success rate. The method has been found to work for a wide variety of genes including a gene located in a telomeric region (lethal giant larvae (l(2)gl)), suggesting that there may be few limitations in its applicability.

To ensure that the expression pattern and protein distribution correspond to the endogenous protein, we costained two tagged lines with GFP for which specific monoclonal antibodies are available: Eyes shut (Eys) (mAb 21A6,) and Delta (Dl) (mAb C594.9B) (Figure 3A). In both cases, the protein recognized by the mAb colocalizes with the GFP and match the described expression patterns (Das et al., 2013; Haltom et al., 2014). Note, however, that the GFP tagged Eys protein is present in the cytoplasm of the photoreceptors and the inter-rhabdomere spaces (IRS) of the photoreceptors, whereas the mAb against Eys mostly localizes to the IRS (Figure 3A). These data are in agreement with what we previously observed for numerous tagged proteins (Venken et al., 2011; Nagarkar-Jaiswal et al., 2015).

Figure 3

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We stained third instar larval brains and discs for the 60 tagged gene/proteins. The examples, shown in Figure 3B, include lethal (2) giant larvae (l(2)gl) (a), Delta (Dl) (b), and twins (tws) (c) whose expression patterns are consistent with published data (Kooh et al., 1993; Albertson and Doe, 2003; Chabu and Doe, 2009). Similarly, kayak/fos (kay) is expressed in wing disc nuclei (d) as described earlier (Zeitlinger and Bohmann, 1999). The expression pattern of the remaining genes has not been previously described (Figure 3B): Saposin-related (Sap-r) is expressed in a subset of cells in larval brain (e), Rad, Gem/Kir family member 3 (Rgk3) is enriched in mushroom body in L3 larval brain (f), Heterogeneous nuclear ribonucleoprotein at 98DE (Hrb98DE) is expressed in L3 larval brain (g), CG10086 is expressed in hindgut (h), and CG5656 is expressed in the cells of the cuticle (i). The expression patterns of all these genes as well as all the genes listed in Supplementary file 1 are documented in the MiMIC RMCE database at http://flypush.imgen.bcm.tmc.edu/pscreen/rmce/.

In summary, we developed a genetic tagging strategy that will greatly facilitate the EGFP tagging of nearly 2000 genes that already carry MiMIC insertions (Nagarkar-Jaiswal et al., 2015). The same strategies can also be used for tagging genes with other protein tags. In addition, a similar strategy based on lox sites instead of FRT cassettes has recently been developed to integrate an artificial exon carrying the GAL4 gene in MiMICs inserted in coding introns (Diao et al., 2015). These insertions are mutagenic but permit the expression of the endogenous wild-type and mutant cDNAs of Drosophila as well as other species under the control of UAS. Moreover, these tagging methods can now be combined with CRISPR directed integration of attP carrying cassettes similar to MiMIC in coding introns to tag almost every gene in Drosophila (Zhang et al., 2014).

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Thank you for submitting your work entitled “A genetic toolkit for tagging intronic MiMIC containing genes” for peer review at eLife. Your submission has been favorably evaluated by K VijayRaghavan (Senior editor), Mani Ramaswami (Reviewing editor), and two reviewers.

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

Summary:

This Research Advance describes the creation of a genetic toolkit that allows for the (Recombination Mediated Cassette Exchange) RMCE of an integrated exon encoding for GFP with existing MIMIC insertions. This resource bypasses the need for laborious injections and will facilitate the use of MIMIC lines for epitope tagging genes at their endogenous sites. An extension of the recent MiMIC resource that was published in eLife, it documents a simplified and streamlined version of converting an intronic MiMIC insertion (containing 2 attP sites) into a GFP tagged protein locus without need for the traditional labour-intensive injection approach. The source of the circular donor plasmid that contains both attB sites and the GFP cassette is a Flp mediated excision of a transgenic insertion. Transgenic lines for all three reading frames on second and third chromosome together with Flp and phiC31 sources were generated. The successful exchange has been demonstrated for 60 loci.

This RMCE approach provides a significant advance of the former MiMIC resource and can also be used beyond MiMIC, e.g. if the attP cassette has been inserted by CRISPR.

Essential revisions:

Several points should be addressed:

1) Figure 1 appears incorrect: surely the white⁺ box should be between the attB site and the FRT.

2) In the paragraph starting with “We observed RMCE events…”, the sentence “We set up 3-7 vials for each starting cross and this was sufficient to obtain 60/116 (113 or 116) tagged genes.” What is meant by (113 or 116)? This paragraph suggests that RMCE is unsuccessful in close to half of the lines tested. Do the authors have any insights into why this may be the case? Is there a common feature amongst the MIMIC lines that failed RMCE?

The authors should provide some idea of how efficient the process is. How many individual events were obtained from the 3-7 starting vials? How many flies were typically screened to get potential recombinants?

3) In “Since the efficiency of RMCE…”, yellow should be replaced by yellow^–.

4) In the same paragraph, the authors should list examples of genes in heterochromatic regions.

5) Given the utility of newly developed CRISPR/Cas9 techniques, the authors should discuss the strengths of MIMIC system. The case can certainly be made. But the GFP cassettes described here represent only an incremental start. This resource would be greatly enhanced by generating lines with additional tags, as previously described by the authors.

1) Figure 1 appears incorrect: surely the white⁺ box should be between the attB site and the FRT.

We thank the reviewers for pointing out this error. We made the correction in Figure 1.

2) In the paragraph starting with “We observed RMCE events…”, the sentence “We set up 3-7 vials for each starting cross and this was sufficient to obtain 60/116 (113 or 116) tagged genes.” What is meant by (113 or 116)? This paragraph suggests that RMCE is unsuccessful in close to half of the lines tested. Do the authors have any insights into why this may be the case? Is there a common feature amongst the MIMIC lines that failed RMCE?

We thank the reviewers for pointing out this issue. We have deleted the numbers.

This paragraph suggests that RMCE is unsuccessful in close to half of the lines tested. Do the authors have any insights into why this may be the case? Is there a common feature amongst the MIMIC lines that failed RMCE? The authors should provide some idea of how efficient the process is.

The RMCE was successful in 82% (93/113) of cases (please see the first sentence of this paragraph). This number includes EGFP cassette insertions in positive and negative orientation. However the probability of getting EGFP inserted in the correct orientation is statistically 50% of total RMCE events. The number 60 is the number of genes for which the EGFP cassette was inserted in the correct orientation resulting in EGFP tagged fusion proteins. Therefore with the current number of crosses (3-7 vials; Cross 2 in Figure 2) the efficiency is 53% (60/113). This can be increased by setting a greater number of screening crosses (10-20 vials per line) to obtain more RMCE events with insertion in positive orientation. Indeed, we recovered significantly more positive events with 7 crosses than with three crosses.

How many individual events were obtained from the 3-7 starting vials? How many flies were typically screened to get potential recombinants?

We obtained 1-10 RMCE positive individuals after screening a total of 300-700 flies from 3-7 vials.

3) In “Since the efficiency of RMCE…”, yellow should be replaced by yellow^–.

We have corrected it.

4) In the same paragraph, the authors should list examples of genes in heterochromatic regions.

We have included the gene name (Lethal giant larva (l(2)gl)) in text.

5) Given the utility of newly developed CRISPR/Cas9 techniques, the authors should discuss the strengths of MIMIC system. The case can certainly be made. But the GFP cassettes described here represent only an incremental start. This resource would be greatly enhanced by generating lines with additional tags, as previously described by the authors.

We have modified the last paragraph of the Discussion.

Author details

1. Sonal Nagarkar-Jaiswal

   Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States

   Contribution

   SN-J, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Steven Z DeLuca

   Competing interests

   The authors declare that no competing interests exist.

2. Steven Z DeLuca

   Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States

   Contribution

   SZDL, Conception and design, Drafting or revising the article

   Contributed equally with

   Sonal Nagarkar-Jaiswal

   Competing interests

   The authors declare that no competing interests exist.

3. Pei-Tseng Lee

   Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   P-TL, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Wen-Wen Lin

   Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   W-WL, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Hongling Pan

   Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States

   Contribution

   HP, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Zhongyuan Zuo

   Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   ZZ, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Jiangxing Lv

   Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   JL, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

8. Allan C Spradling

   Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States

   Contribution

   ACS, Conception and design, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

9. Hugo J Bellen

   1. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States
   2. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
   3. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
   4. Department of Neuroscience, Baylor College of Medicine, Houston, United States
   5. Program in Developmental Biology, Baylor College of Medicine, Houston, United States

   Contribution

   HJB, Conception and design, Drafting or revising the article

   For correspondence

   hbellen@bcm.edu

   Competing interests

   The authors declare that no competing interests exist.

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