Fig. 2

Knock-in of a conditional Cas9 transgene into Rosa26 of C57BL/6 zygotes. a: Strategy for insertion of the CAG-loxPSTOPloxP-Cas9-IRES-EGFP cassette into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 4 kb fragments used as homology arms in the targeting vector. Homology-directed repair (HDR) leads to the insertion of the cassette into the genome. The locations of PCR primers, restriction sites and the Rosa26 hybridisation probe in the targeted and wildtype alleles are indicated. b: Gel electrophoresis of XbaI digested Rosa26 PCR products (R26F2/R2 primers) amplified from pups (#7-#46) derived from microinjections of targeting vector, sgRosa26-1 and Cas9 RNAs. 0.2 kb bands of XbaI resistant products (mut) indicate sequence deletions, wildtype products (wt) are reduced to 0.1 kb. M - size marker, B6 - C57BL/6 wildtype control. c: PCR detection of an internal segment of Cas9 in pups derived from microinjections using primers Cas9F/Cas9R (top). Bottom: three primer PCR for the simultaneous detection of the Rosa26 target region (R26F2/R2 primers, 0.2 kb) and of vector sequences (R26F2-SAR, 0.12 kb), showing that all samples harbor at least one nonrecombined Rosa26 allele. V – vector positive control, H₂O – negative control. d: Cas9-positive mice (from b) were further tested for correct knock-in (KI) into Rosa26 using a PCR reaction with a forward primer located outside of the 5′-homology region (R26F3) and a reverse primer located in transgene (SAR); the predicted band has a size of 1.38 kb (top). Bottom: DNA quality was controlled with a Cas9 internal PCR (Cas9F/R primers,0.38 kb). H₂O – negative control. e: Southern blot analysis of EcoRI digested tail DNA from Cas9-positive mice (from b) using an external Rosa26-specific hybridization. Knock-in alleles are predicted to show a 6 kb band. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control. f: Genotyping PCR of 15 F1 pups derived from founder mutants #35 or #39 using the Cas9 internal primer pair Cas9F/R. g: Southern blot analysis of EcoRI digested tail DNA from two F1 pups using an external Rosa26-specific hybridization probe. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control