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Loss of Nectin-2 at Sertoli-Spermatid: MATERIALS AND METHODS(2)

Fragment 1 (2.2 kilobases [kb]), spanning the C-terminal portion of exon 3, intron 3, and the N-terminal portion of exon 4, was inserted into the SalI XhdL sites of pBS-ntz, resulting in vector pBS-ntz-2k. To generate the final targeting vector, pMPH-ko, fragment 2 (10 kb), spanning exon 1, intron 1, and the N-terminal part of exon 2, was inserted upstream and in frame with the LacZ ORF, into the NotVAscl sites of pBS-ntz-2k. Thus, on successful homologous recombination, the mutated nectin-2 locus will express a chimeric reporter protein of the first 61 codons of nectin-2 fused to LacZ. In addition, the targeted locus has lost most of exons 2 and 3 (Fig. 1). The procedure, leading to the knockout of a functional nectin-2 gene, resulted in the knockin of the LacZ gene. We will refer to genetically altered, homozygous animals as nectin-2LacZ/LacZmice.

The targeting vector was electroporated into embryonic stem (ES) cells derived from male 129Sv/J blastomeres (Genome Systems, Inc., St. Louis, MO). The ES clones were selected through a positive/negative selection procedure with G418 and gancyclovir. Targeted ES clones were identified by genomic PCR with oligonucleotides pgkpA(+)/Ex5(—), yielding a 3.5kb reaction product, whereas random insertions do not produce a PCR product (Fig. 1B). Positive ES cell clones were confirmed by Southern blot analysis of NdeI (New England Biolabs)-digested genomic DNA (Fig. 1C).