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Loss of Nectin-2 at Sertoli-Spermatid: MATERIALS AND METHODS(1)

METHODS(1)

Generation of Nectin-2 Knockout Mice

A gene targeting vector, pMPH-ko, was constructed in which a selectable pgk-neo cassette is flanked by two genomic fragments of the murine nectin-2 gene (Fig. 1). In detail, a HindIII/EcoRI fragment containing the pgk-neo cassette of pKJ1 was subcloned into the HindIII/EcoRI sites of pBS-SK(+) (Stratagene, La Jolla, CA) to generate pBS-pgkneo. A polymerase chain reaction (PCR) fragment (oligonucleotides LacZ(+)/pUCRI(—)) containing the open reading frame (ORF) of Escherichia coli p-galactosidase (LacZ) and a SV40 poly(A) signal was amplified from vector pSDK.LacZpA128 (J. Rossant, University of Toronto, Toronto, Canada) and inserted into Xbal/Xmal sites of pBS-pgkneo.

The LacZ-pgkneo cassette was then liberated using XhoI/XbaI and inserted into the Xhol/Xbal linearized vector pBltk4 containing the thymidine kinase gene of herpes simplex virus-1 (HSV-1) under its endogenous viral gene promoter. This allowed for negative selection against random insertion events of the final targeting vector. Into the resulting vector, pBS-ntz, two nectin-2 gene fragments were inserted. These fragments were generated by PCR from 129Sv/J genomic DNA using the LA-PCR kit (Takara, Shiga, Japan) in combination with oligonucleotide pairs Ex3(+)/Ex4(—) and Ex1(+)/Ex2(—), respectively.