A genetic toolkit for tagging intronic MiMIC containing genes

1One 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. 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. However, previous methods for generating protein trap alleles in Drosophila have allowed only about 800 genes to be successfully tagged.

2We 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. 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, and MiMIC-like elements can now be placed in any gene of interest by CRISPR. 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.

3We 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. 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. We then cloned this cassette core between tandem FRT sites in a P-element transformation vector. 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. 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.

4We 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.

5To 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. Typically, 50% of the integration events are in the proper orientation.

6We 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.

7To 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. 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.

8We 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. Similarly, kayak/fos (kay) is expressed in wing disc nuclei (d) as described earlier. 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/.

9In summary, we developed a genetic tagging strategy that will greatly facilitate the EGFP tagging of nearly 2000 genes that already carry MiMIC insertions. 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. 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.

10The core cassettes (attB-SA- phase 0/1/2-[GGS]4-EGFP-FlAsH-StrepII-TEVcs-3xFlag-[GGS]4-SD-attB) for Phase 0,1, or 2 were excised from pBS-KS-attB1-2-PT-SA-SD-0-EGFP-FlAsH-StrepII-TEVcs-3xFlag, pBS-KS-attB1-2-PT-SA-SD-1-EGFP-FlAsH-StrepII-TEVcs-3xFlag, or pBS-KS-attB1-2-PT-SA-SD-2-EGFP-FlAsH-StrepII-TEVcs-3xFlag as NheI/NsiI fragments and subcloned into P-element vector pW35 (DGRC) between PstI/AvrII to create final donor vectors pW35-FRT-attB-SA-phase 0-(GGS)4-EGFP-FlAsH-StrepII-TEVcs-3xFlag-(GGS)4-SD-attB-white+-FRT, pW35-FRT-attB-SA-phase 1-(GGS)4-EGFP-FlAsH-StrepII-TEVcs-3xFlag-(GGS)4-SD-attB-white+-FRT and pW35-FRT-attB-SA-phase 2-(GGS)4-EGFP-FlAsH-StrepII-TEVcs-3xFlag-(GGS)4-SD-attB-white+-FRT.

11Around 15–20 phase-specific homozygous donor females (P{ry+t7.2= hsFLP}12, y1w*M{vas-int.B}ZH-2A; P{FRT-attB-{GFSTF}-attB(w+)-FRT}; Pri1/TM6B, Tb1 or P{ry+t7.2 = hsFLP}12, y1 w*M{vas-int.B}ZH-2A; S1/CyO; P{FRT-attB-{GFSTF}-attB (w+)-FRT}) were crossed with 5–10 males carrying MiMIC insertion in coding intron (y1 w*; Mi[MIC y+] geneMI/Mi[MIC y+] geneMI or balancer). Crosses were transferred to new vials every third day and constantly kept at 18°C. Vials with progeny embryos and larvae were heat shocked on day 3, 4, and 5 for 20 minutes at 37°C in a water bath and raised at 25°C. About 5–7 vials with a pool of 5 F1 males with mosaic red eyes and yellow body were crossed with 10–15 virgins of y1w67c23; In(2LR)Gla, wgGla−1/SM6a or y1w*; D/TM6b, Hu, Tb. Transgenic F2 progeny were screened for loss of yellow+ (yellow-phenotype) and subsequently crossed to virgins of y1w67c23; In(2LR)Gla, wgGla−1/SM6a or y1w*; D/TM3,Sb, Tb to establish stocks. Correct RMCE events were confirmed by PCR assay as described in.

12Briefly, third instar larvae were dissected for larval brains, imaginal discs, and gut in 1xPBS and fixed in 3.7% formaldehyde for 30 min at room temperature and washed in 0.2% Triton X-100. They were then incubated for 1 hr at RT in 10% NGS-PBS-0.2% Triton X-100 and stained with primary antibodies diluted in 10% NGS-PBS-0.2% Triton X-100 overnight at 4°C. The samples were washed and incubated with secondary antibodies for 2 hr at RT. The samples were then washed and mounted in Vectashield (Vector Labs, Burlingame, CA) and imaged with a Zeiss LSM710 confocal microscope and processed using Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA).

13Primary antibodies used: rabbit anti-GFP 1:1000 (Life Technologies, A11122), mouse anti-Delta 1:1000 (C594.9B, DSHB), and mouse anti-Eys 1:250 (21A6, DSHB). Secondary antibodies used: Alexa 488 (Invitrogen, Life Technologies, Grand Island, NY), Cy5 and Cy3 conjugated antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:500.