1. Chromosomes and Gene Expression
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A gene-specific T2A-GAL4 library for Drosophila

  1. Pei-Tseng Lee
  2. Jonathan Zirin
  3. Oguz Kanca
  4. Wen-Wen Lin
  5. Karen L Schulze
  6. David Li-Kroeger
  7. Rong Tao
  8. Colby Devereaux
  9. Yanhui Hu
  10. Verena Chung
  11. Ying Fang
  12. Yuchun He
  13. Hongling Pan
  14. Ming Ge
  15. Zhongyuan Zuo
  16. Benjamin E Housden
  17. Stephanie E Mohr
  18. Shinya Yamamoto
  19. Robert W Levis
  20. Allan C Spradling
  21. Norbert Perrimon
  22. Hugo J Bellen  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Harvard Medical School, United States
  3. Texas Children’s Hospital, United States
  4. Carnegie Institution for Science, United States
Tools and Resources
Cite this article as: eLife 2018;7:e35574 doi: 10.7554/eLife.35574
5 figures, 1 table and 4 additional files

Figures

Figure 1 with 2 supplements
Protein distribution and expression patterns of genes containing MiMICs tagged with GFSTF or T2A-GAL4.

The MiMIC transposon contains two inverted attP sites that allow RMCE. (A) Detection of the expression domains of the indicated genes tagged with GFSTF in larvae and adult brains. GFP: green (B) Schematic of the MiMIC conversion with Trojan triplet T2A-GAL4 cassettes (Diao et al., 2015). Only the inserted T2A-GAL4 cassette with the correct orientation and phase results in GAL4 expression that drives UAS-mCD8::GFP expression. (C) Detection of the expression domains in larvae and adult brains of genes tagged with T2A-GAL4 using UAS-mCD8::GFP. mCD8::GFP: green. Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.003
Figure 1—figure supplement 1
Expression of MiMIC GFSTFs tagged genes in adult brains.

We document the expression patterns and protein localization associated with 49 genes. The MiMIC identification number and affected genes are shown. Note that only ~19% of all genes tested showed detectable GFP signals in adult brains. Images are available on Flypush (http://flypush.imgen.bcm.tmc.edu/pscreen/rmce/). Many patterns are in agreement with the expression patterns of published genes/proteins (e.g. brp, Rgk1, dlg1, cact). Green: GFSTF. Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.004
Figure 1—figure supplement 2
Similarities and differences between expression patterns associated with GAL4 >UAS GFP driven patterns and endogenous proteins in adult brains.

(A) Comparison of T2A-GAL4 > UAS-mCD8::GFP with anti-body stainings for arm (larval wing disc), Mhc (larval muscle), osa (larval eye-antennal imaginal disc) and trio (larval CNS and boutons/NMJ). Green: mCD8::GFP, Red: antibody labeling. (B) Comparison of T2A-GAL4 > UAS-mCD8::GFP with GFSTF for SIFaR, zip, VGlut and mbl in adult brains. Note the striking differences. Green in left panel: mCD8::GFP; Green in right panel: GFSTF. Scale bar is shown in figure.

https://doi.org/10.7554/eLife.35574.005
Figure 2 with 1 supplement
CRIMIC pM14 and pM36, and Golden Gate Assembly.

(A) Structures of pM14 and pM36. The CRIMIC pM14 cassette contains MiMIC-like cassette (SA-3xstop-polyA) and two FRT sites. The CRIMIC pM36 cassette was modified by removing the two FRT sites from PM14. (B) Golden Gate Assembly. Two sets of primers containing Type IIS RE sites are typically used to amplify ~1 kb homology arms by PCR. These arms, pM37 DNA and pBH vector (KanR) digested with Type IIS Restriction Enzymes and cloned using Golden Gate Assembly to generate the donor construct in a single reaction. The pM14/pM36 based donor DNAs were constructed with the same approach. The complete donor construct is selected with kanamycin. The components in these diagrams are not drawn to scale.

https://doi.org/10.7554/eLife.35574.006
Figure 2—figure supplement 1
The efficiency of cassette insertion with CRIMIC pM14 and pM36, and PCR validation.

(A) Summary of the microinjection success rates for pM14 and pM36. We injected embryos for 89 genes with pM14 and the success rate was 57.3%. We also injected embryos for 114 genes with pM36 and the success rate was 26.3%. (B) The CRIMIC flies were verified by PCR to ensure that cassettes are in the correct position. Two specific primers outside homology arms (gene-F/gene-R) in each gene were designed to amplify the homologous arm regions by PCR with attP-R primer. The CR00711 (slim T2A-GAL4) and CR00684 (boca T2A-GAL4) are shown here. The genomic DNA of individual alleles was extracted and PCR was performed (see Materials and methods). The #2, #3 and #6 of slim T2A-GAL4s are correct alleles, and #2 and #3 of boca T2A-GAL4s are correct alleles. L: left region of the inserted cassette; R: right region of the inserted cassette.

https://doi.org/10.7554/eLife.35574.007
CRIMIC: T2A-GAL4 integration using CRISPR and expression patterns of tagged genes.

(A) Structure of the CRIMIC pM37 cassette. (B) Schematic of the CRIMIC insertion strategy through two 1 kb homology arms by HDR (homology directed repair) based on CRISPR/Cas9 technology. (C) Expression patterns observed in adult fly brains of T2A-GAL4 > UAS-mCD8::RFP. mCD8::RFP (red). Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.008
Figure 4 with 2 supplements
Summary of CRIMIC T2-GAL4 integration efficiency and genetic properties of T2A-GAL4 insertions (A) microinjection success rates for pM37.

(B) Complementation test: 90% of the T2A-GAL4 containing chromosomes fail to complement the corresponding Dfs; 5% produced less than 1/3 of the expected progeny; and 5% fully complemented the Dfs. For details see Supplemental Information 2. (C) T2A-GAL4 cassette excision. The lethality associated with 8 out 11 insertions is reverted in the presence of UAS-FLP. (D) Rescue of the lethality of the T2A-GAL4 cassette insertions with UAS-cDNA.

https://doi.org/10.7554/eLife.35574.009
Figure 4—figure supplement 1
Applications of the CRIMIC technology.

(A) The CRIMIC pM37 cassette contains polyA 3’ of GAL4 which will arrest transcription and typically generate a severe loss-of-function allele. (B) The lethality caused by SA-T2A-GAL4-polyA cassette insertion can often be reverted by cassette excision using the FRT sites at the ends of the cassette and FLP. (C) The inserted SA-T2A-GAL4-polyA cassette can be replaced with other DNA fragments containing two attB sites and phiC31 integrase by RMCE such as GFSTF. (D) The SA-T2A-GAL4-polyA insertion produces GAL4 that can be used to rescue the phenotype (lethality caused by the cassette insertion) with UAS-cDNA.

https://doi.org/10.7554/eLife.35574.010
Figure 4—figure supplement 2
T2A-GAL4 cassette excision upon FRT-FLP.

CRIMIC T2A-GAL4 cassette, pM37 contains two FRT sites. The cassettes in three genes (Dsor1, Raf, Marf) failed to revert the lethality when exposed to UAS-FLP. However, cassette excision is still effective based on the loss of the 3XP3 Eye-GFP in Dsor1; however, weak GFP expression remains in Raf and Marf. Left panel: CRIMIC T2A-GAL4s; Right panel: CRIMIC T2A-GAL4s > UAS FLP. Scale bar: 0.2 mm.

https://doi.org/10.7554/eLife.35574.011
Figure 5 with 3 supplements
Genes expressed in (A) trachea, (B) glial cells, and (C) Pars Intercerebralis Neurons based on T2-GAL4 insertions.

The GAL4s (underlined) are existing P-element enhancer traps expressing GAL4 in specific cell populations and serve as controls. mCD8::GFP: green. Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.013
Figure 5—figure supplement 1
Genes specifically expressed in trachea.

Five genes previously not reported to be expressed in trachea (sprt, geko, ex, Samuel, Cad96Ca). Expression in trachea in adult brains and VNC. MiMIC T2A-GAL4s were crossed with UAS-mCD8::GFP (green). Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.014
Figure 5—figure supplement 2
Genes expressed in glia.

Two genes (Egfr, Tret1-1) previously documented to be expressed in glia and twelve genes not previously documented to be expressed in glia (bdl, Zasp52, rols, ine, CG5404, CG14688, CG31663, ry, CG4752,βTub97EF, CG32473, LManII) in adult brains. MiMIC T2A- GAL4s were crossed with UAS-mCD8::GFP (green). Note that the damages were caused by dissections in MI02852-Egfr and MI02136-ry. Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.015
Figure 5—figure supplement 3
Genes expressed in PI neurons.

16 genes (NimB2, Lerp, CG7744, cnc, CG2656, spin, gem, Fs, Aldh-III, CG33056, grsm, CG31075, Pi3K68D, Dh44-R2, Lgr4, Atg16) exhibit relatively specific expression in PI neurons in adult brains. MiMIC T2A-GAL4s were crossed with UAS-mCD8::GFP (green). Scale bar: 50 μm.

https://doi.org/10.7554/eLife.35574.016

Tables

Table 1
Rescue of the lethality of T2A-GAL4s insertions/Dfs with aUAS-cDNA and genomic duplications with P[acman] clones.

*1:(Luo et al., 2017)*2:(Chao et al., 2017)*3:(Yoon et al., 2017)*4:(Sandoval et al., 2014). Note that a failure to rescue lethality does not mean that it cannot partially rescue other scorable phenotypes.

https://doi.org/10.7554/eLife.35574.012
Flies for rescue
LineGeneChr.Protein isoformsFlies for complementation testFly cDNAGenomic DNA
MI01374-TG4.0sbrX1NAno tagDp(1;3)DC508
MI02836-TG4.0cac*1X8NAEGFPDp(1;3)DC131
MI07818-TG4.0acj6X13NA3xHADp(1;3)DC192
MI08675-TG4.1armX2NA3xHADp(1;3)DC034
MI10323-TG4.1flwX2NA1xHADp(1;3)DC224
MI12214-TG4.2ifX2NAno tagDp(1;3)DC319
MI00783-TG4.0stj23Df(2R)Exel7128/CyO3xHANA
MI02963-TG4.0CAP220Df(2R)BSC281/CyOno tagNA
MI03306-TG4.1kuz24Df(2L)BSC147/CyOno tagNA
MI03597-TG4.1mol22Df(2R)Exel6066/CyO3xHANA
MI04800-TG4.1lola220Df(2R)ED2076/SM6a3xHANA
MI06876-TG4.1spin23Df(2R)Jp8, w[+]/CyOmyc-EGFPNA
MI09180-TG4.1Bsg22Df(2L)ED548/SM6a3xHANA
MI09585-TG4.1Lpt22Df(2R)BSC610/SM6a1xHANA
MI13162-TG4.0Rho121Df(2R)ED2457/SM6a3xHANA
MI13708-TG4.0Cka24P{ry[+t7.2]=PZ}Cka[05836] cn[1]/CyOEGFPNA
MI15480-TG4.2kn*225Df(2R)BSC429/CyO3xHANA
MI02220-TG4.1dally31Df(3L)ED4413/TM6C, cu[1] Sb[1]no tagNA
MI04910-TG4.1ftz-f133Df(3L)BSC844/TM6C, Sb[1] cu[1]3xHANA
MI06026-TG4.1Nc73EF*333Df(3L)ED4685/TM6C,cu[1] Sb[1]FlagDp(1;3)DC245
MI07056-TG4.0Atg132Df(3L)BSC613/TM6C, cu[1] Sb[1]no tagNA
MI08143-TG4.0Sod132Df(3L)BSC817/TM6C, Sb[1] cu[1]no tagNA
MI05068-TG4.0kdnX2NANADp(1;3)DC154
LineGeneChr.TranscriptsDfFly cDNAGenomic DNA
CR00323MarfX2NA1xHA*4Dp(1;3)DC155
CR00446Dsor1X2NA3xHADp(1;3)DC205
CR00483RafX1NAno tagDp(1;3)DC404
CR00505RbfX1NA3xHADp(1;3)DC012
CR00638MoeX7NAmycDp(1;3)DC199
CR00354sax23Df(2R)BSC265/CyO3xHANA
CR00465Dap16026Df(2L)BSC302/CyOno tagNA
CR00466Eps-1524Df(2R)BSC606/SM6ano tagNA
CR00494l(2)gd122Df(2L)Exel6027/CyO1xHANA
CR00521Npc1a22Df(2L)BSC143/CyOYFPNA
CR00559Sod221Df(2R)Exel7145/CyOno tagNA
CR00587Hr3822Df(3R)BSC510/TM6C, Sb[1] cu[1]3xHANA
CR00762Wee121Df(2L)BSC108/CyOno tagNA
CR00452sr34Df(3R)BSC510/TM6C, Sb[1] cu[1]no tagNA
  1. Blue: fail to complement

    Gray: partially complement

  2. Green: rescued

    Pink: fail to rescue

  3. Orange: rescue phenotype but not lethality

Additional files

Supplementary file 1

CRISPR crosses.

https://doi.org/10.7554/eLife.35574.017
Supplementary file 2

Information of MiMIC/CRIMIC lines, fly stocks, complementation results and resource.

https://doi.org/10.7554/eLife.35574.018
Supplementary file 3

DNA sequences of CRIMIC donor vectors.

https://doi.org/10.7554/eLife.35574.019
Transparent reporting form
https://doi.org/10.7554/eLife.35574.020

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