Molecular analysis of F1 GeneWeld GTag targeted alleles at tyr and noto.
(a–c) Molecular analysis of Tg(tyr-2A-GAL4/VP16) F1 offspring from a single targeted F0 founder. (a) Schematic of expected integration pattern for tyr targeted with pGTag-2A-GAL4/VP16. 148 bp tyr probe in Exon 3 and 583 bp probe in GAL4/VP16 are indicated. (b) GAL4/VP16 and (c) tyr probed Southern blots of genomic DNA from wild type (WIK) and 4 individual GAL4/VP16 positive F1s. The expected 7400 bp band is detected with both probes, suggesting a single copy integration. (d–f) Tg(noto-2A-RFP) F1 targeted integration alleles from 2 independent F0 founders. (d) noto gene model with location of restriction enzymes used for genomic Southern blot analysis. Location of the 513 bp noto probe is indicated (dark lines). The predicted and an interpretation of the recovered alleles are shown. (e) Southern blots of F1 Tg(noto-2A-RFP) individuals hybridized with RFP probe. F1 from founder F0#1 contain a ~ 2100 bp band corresponding to integration plus deletion of ~400 bp in noto. F1 progeny from founder F0#2 show two bands: a ~ 3700 bp band corresponding to integration of the reporter plus 2000 bp of vector backbone, and a ~ 1500 bp band which may represent an off-target integration. Loading controls (10, 1) correspond to 10 copies or 1 copy of RFP containing plasmid. WIK, wild type control DNA. (f) Southern blot in (e) stripped and re-hybridized with the noto-specific probe. A 1342 bp band representing the wild type allele was detected in all individuals. The integration allele in F1s from F0 #1 was not detected due to deletion of the region containing the probe. F1s from F0 #2 contain the ~3700 bp band corresponding to the noto-2A-RFP integration allele.
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