Fig. 5

Insertion and selection of the NRAMP1 transgenic colony using single Cas9 or Cas9n. a Schematic representation of the gene-targeting donor vector. b Schematic overview of the screening of the individual colonies. Lj F and Rj R were the primers for the regions outside the homologous arms, and Lj R and Rj F were the primers for the donor vector region. Southern blot probes are shown as red lines and Hind III digestion was used in the southern blot analysis. c Sanger sequencing confirming the precise insertion of the exogenous DNA. d Southern blot analysis of the donor cells used for SCNT. Non-transfected BFFs were used for negative controls. A 1.58 kb band resulting from the targeted insertion of the NRAMP1 cassette was detected in addition to the 3.84 kb band from the endogenous F-A locus allele when probe 1 was used. A 9.05 kb targeted band was also detected with probe 2