Targeting of the Nectin-2 Gene Locus
We have inactivated the nectin-2 gene locus by homologous recombination in mouse ES cells of the 129Sv/J genetic background. A targeting vector was constructed that on successful recombination replaces most of exons 2 and 3 by an in-frame fusion with the LacZ ORF and a selectable pgk-neo cassette (Fig. 1). Thus, a fusion protein between the N-terminal 61 amino acids of nectin-2 and LacZ is expressed under control of the endogenous nectin-2 gene promoter. Of 355 ES cell clones screened, 8 were found to contain the targeted nectin-2 gene locus. Two independent screening procedures were used to confirm successful targeting. First, a primary PCR screen with oligonucleotide pair pgkpA(+)/Ex5 was employed that identified positive clones by the presence of 3.5-kb reaction product. other
Because the annealing site for Ex5 is located outside the replacement cassette, only homologous recombination events bring the two annealing sites into proximity to result in an ampli-con of the predicted size. Consequently, random insertions should not produce a PCR product (Fig. 1B). Clones identified by this method were then subjected to Southern blot analysis of AWeI-digested genomic DNA. An external probe was chosen that comprises exons 5 and 6. This probe was amplified from nectin-2 cDNA and subsequently 32P-labeled by random priming. Whereas a wt allele produces a band of approximately 13 kb, the targeted allele is characterized by a 16-kb band (Fig. 1C). All clones identified in the primary PCR screen yielded the characteristic doublet of bands (Fig. 1C).
FIG. 1. Generation of nectin-2 knockout mice. A) Construction of the gene targeting vector. At the top is the organization of the nectin-2 gene locus (after Morri son and Racan i ello ). S i x exons contri bute to the ORF. On the ri ght, a schemati c of the doma i n structure of necti n-2 at the prote i n level is presented. Three extracellular immunoglobulin-like domains are followed by a transmembrane domain (T) and a cytoplasmic tail. In the middle is the gene targeting construct pMPH-k.o. A selectable pgk-neo cassette is flanked by two nectin-2 gene fragments. The ORF of LacZ is fused in frame to exon 61 of nectin-2. An external HSV-1 thymidine kinase (tk) gene allows for negative selection. At the bottom is the mutated nectin-2 locus after homologous recombination. Most of exons 2 and 3 and the intervening intron are deleted and replaced by the pgk-neo cassette. The LacZ reporter is expressed as a fusion to the first 61 amino acids of nectin-2 (schematic on the right) under control of the endogenous nectin-2 gene promoter. Bold lines and black boxes represent genomic sequences. B) Primary PCR screen of ES clones. A 3.5-kb PCR product (lanes 3, 4, and 10) results only from clones that have undergone homologous recombination, because downstream primer Ex5(—) is located outside the replacement cassette (see Materials and Methods). C) Clone confirmation by Southern blot analysis. Clones identified by PCR were tested by Southern blotting of NdeI-digested DNA with an external probe comprising the cDNA sequences of exons 5 and 6 (538 bp). In the targeted allele, the wt-specific, 13-kb band is replaced by a 16kb band. D) PCR genotyping of mouse offspring by three-primer PCR. A 210-bp PCR product spanning the nectin-2-LacZ junction indicates the mutant allele, whereas a 350-bp amplification product of intact exon 2 identifies the wt allele. Thus, in heterozygous animals, both bands are amplified (lanes 2, 4, 7, and 9), but in wt or homozygous mutants, only the 350-bp amplicon (lanes 3, 5, and 6) or the 210-bp amplicon (lane 8), respectively, is seen.