Generation of a knockin GFP-CRE allele at the <i>Wisp3</i> locus.

<p>(Upper panel) Schematic diagram of the <i>Wisp3</i> locus, the targeting vector, and the <i>Wisp3</i>^lacZΔex3-5 and <i>Wisp3</i>^{GFP- Cre} alleles (not drawn to scale). Exons 1-5 are indicated as rectangles, with the 5’ untranslated region (UTR) of exon 1 a thinner rectangle. The <i>Wisp3</i>^GFP-Cre targeting construct contains a thymidine kinase cassette (TK) for negative selection, an enhanced Green Fluorescent Protein (GFP) cassette, an internal ribosomal entry site (IRES), a Cre-recombinase (Cre) cassette, a polyA addition signal (rGpA), and a FRT (diamonds) flanked Neomycin resistance cassette (NeoR), all inserted into <i>Wisp3</i> exon 1. DNA sequences from the correctly targeted <i>Wisp3</i>^GFP-Cre allele are shown. The two potential translation initiation codons of endogenous <i>Wisp3</i> are underlined, as is the translation initiation codon in the GFP expression cassette. NotI and SalI restriction sites are in italics. FRT sequence 3’ of the NeoR cassette, the remainder of <i>Wisp3</i> exon 1, and beginning of <i>Wisp3</i> intron 1 are indicated. Arrows indicate the locations of the PCR primers used to generate the amplimers shown in the lower panels. The P1 and P4 primers lie outside of the targeting arms (Lower panels). A) PCR amplimers demonstrating correctly targeted alleles in <i>Wisp3</i>^{GFP-Cre/GFP-Cre} mice and their absence in wild-type mice. B) PCR amplimers demonstrating insertion of the GFP-IRES-Cre cassette into <i>Wisp3</i> exon 1 in heterozygous and homozygous knockin mice (arrows indicate the expected products from the knockin and wild-type alleles). C) PCR amplimers used to genotype wild-type, heterozygous, and homozygous knockin littermates and the <i>Wisp3</i> locus in C57BL6/J mice (control). Sizes of the molecular weight standards (MS Std) in kb or bp are indicated.