Fig. 2: Confirmation of the transgene insertion site on Chromosome 7, and genotyping of zygosity.

a Map of the transgene/chromosome 7 left and right junctions. Primers were designed to confirm the left and right junctions between the transgene and Chromosome 7, as well as the predicted deletion. b Primers 1 and 2 were used to confirm the 27 kb deletion. The 250 bp band is absent in putative homozygous α-Cre animals. Primers 1 and 5 were used to confirm the left junction between the 3′ end of the α-Cre transgene and the 5′ end of Chromosome 7 in homozygous animals. This 722 bp band is absent in wild-type animals. c The chromatogram of the 722 bp band in b showing the chromosome 7 sequence linked to the Hbb intron through an 18 bp linker. No intervening SV40 poly A sequence was detected. d Primers 3 and 4 were designed to confirm the right-hand junction. However, the 250 bp band is still present in putative homozygous α-Cre animals (arrow). Primers 4 and 6 were used to confirm the right junction between the 5′ end of the α-Cre transgene and the 3′ end of Chromosome 7 in homozygous animals. This 436 bp band is absent in wild-type animals. e The 500 bp chromosome 7 sequence at the right junction of the α-Cre transgene is repetitive (version Mm10). Four such repeats are indicated around Vmn2r67 and Vmn2r68, (red arrows). f Genotyping zygosity of the α-Cre transgene, using primers 1 + 2 + 5 from the left junction. Totally 7 animals were genotyped, including 2 wild types, 2 hemizygotes, and 3 homozygotes.