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. Author manuscript; available in PMC: 2018 Nov 13.
Published in final edited form as: Adv Exp Med Biol. 2018;1066:141–185. doi: 10.1007/978-3-319-89512-3_8

Figure 5: Strategies to “humanize” Drosophila genes in vivo.

Figure 5:

A) For genes that have coding introns (introns flanking two coding exons), one can insert a T2A-GAL4 cassette via CRISPR and HDR (homology directed repair). When the gene of interest is translated, the splice acceptor (SA) forces the splicing machinery to include the T2A-GAL4 cassette. The transcriptional termination site (polyA) stops the transcription, preventing the rest of the gene to be transcribed. When the transcript (mRNA) is translated, N-terminal of the fly protein is made but is prematurely truncated due to the T2A (2A) ribosomal skipping sequence, leading to generation of nonfunctional proteins in most cases. T2A sequence further allows the GAL4 protein to be translated, which in turn translocates to the nucleus to activate the expression of human cDNAs (wild-type/reference or mutant/variant) under the control of UAS elements. B) For genes that do not have a coding intron, one can knock-in a GAL4 in the fly gene of interest. GAL4 will be transcribed and translated in the same temporal and special manner as the fly gene, allowing one to express the human cDNA in a mutant background. Grey boxes: 5’ and 3’ untranslated regions. Orange box: Fly coding sequence (CDS).