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. 2018 Mar 22;7:e35574. doi: 10.7554/eLife.35574

Figure 4. 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.

Figure 4.

Figure 4—figure supplement 1. Applications of the CRIMIC technology.

Figure 4—figure supplement 1.

(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.
Figure 4—figure supplement 2. T2A-GAL4 cassette excision upon FRT-FLP.

Figure 4—figure supplement 2.

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.