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. 2016 Dec 22;100(1):128–137. doi: 10.1016/j.ajhg.2016.11.018

Figure 3.

Figure 3

Generation and Characterization of the Fly kn-T2A-GAL4 Allele

(A) High conservation of protein structure and amino acid sequence between human EBF3 and fly Knot with 62% identity and 70% similarity. Conserved domains include the DNA-binding domain (DBD, red), Zn2+ finger COE motif (pink), Ig-like/plexins/transcription factors (IPT) domain (cyan), helix-loop-helix dimerization motif with α helices H1 (green) and H2 or H2′ (blue), and C-terminal transactivation domain (yellow).

(B) Conversion of the MI15480 line with a MiMIC transposable-element insertion in the fourth coding intron of knot via ϕC31-mediated RMCE for generation of the kn-T2A-GAL4 allele, which expresses GAL4 transactivator in the pattern of kn. This allele also creates a loss-of-function allele of knot by prematurely truncating the transcript by a ribosomal skipping signal (T2A) and a premature polyadenylation signal (pA).

(C) Complementation testing shows that kn-T2A-GAL4 with both the amorphic kncol-1 and genomic deficiency Df(2R)BSC429, encompassing the entire kn locus, fails to complement the lethality. Complementation with hypomorphic kn1 is semi-lethal with <10% viability.

(D) For expression analysis, yw/y; kn-T2A-GAL4/CyO, Kr-GAL4, UAS-GFP males were crossed with yw; UAS-cDNA/TM3 Sb, Kr-GAL4, UAS-GFP virgin females, and double heterozygotes were selected by loss of GFP expression. Images were acquired on a Leica Sp8 laser-scanning confocal microscope. The same settings for laser power and detector gain were used for all genotypes. Images were acquired as a z stack with a z-step of 1 μm and line average of 4 at 400 Hz with a 25× water objective at 1024 × 1024 pixel resolution. Maximum intensity projections were created from the stack in ImageJ. Immunolabeling revealed that kn-T2A-GAL4 recapitulates the endogenous knot expression pattern in third instar wing disc, as shown with the HA-tagged UAS fly lines for WT Knot, WT EBF3, and the EBF3 p.Arg163Gln and p.Arg163Leu variants. Images show HA (magenta) and DAPI (gray). The scale bar represents 100 μm.