Fig. 1.

Generation of Ins2^Apple.LCA mice. A: schematic of the Ins2.Apple.LCA targeting vector, which contains a Lox66 site, the histone 2B (H2B)-Apple sequence, an FRT-flanked puromycin resistance-Δ-thymidine kinase (PU-ΔTK) selection marker, and a Lox2272 site. Ins2^WT represents the wild-type Ins2 allele. The Ins2^LCA allele was created by homologous recombination in mouse embryonic stem (ES) cells. To generate the final allele, mice expressing the Ins2^Apple.LCA allele were crossed with mice expressing FLPe to excise the PU-ΔTK cassette. Primer binding sites are represented by arrows above the schemes. DT-A, diphtheria toxin A; LA, long homology arm; SA, short homology arm. B: Southern blot analyses using either BsrGI-digested ES cell clone DNA and the 5′-probe or NsiI-digested ES cell clone DNA and the 3′-probe. Clone 1C4 was injected into mouse blastocysts to generate live mice. TM, targeted mutation; WT, wild-type. C: PCR analysis used to distinguish between the wild-type and targeted Ins2 alleles.