Construction of MCAD targeting vector.
A neomycin resistance gene cassette [23] was subcloned into the SalI site of pGEM11Zf(+). The plasmid was digested with EcoRI and the overhangs were filled with Klenow enzyme. Subsequent ligation of the blunt ends recircularized the vector and destroyed the EcoRI site within the polylinker of the pGEMl1Zf(+) plasmid. Next, an 8-kb Acadm genomic fragment spanning exons 8, 9, and 10 and flanking intron sequences, originally obtained from a Lambda FIXII 129Sv mouse genomic library [24,25], was directionally cloned into the NotI and XhoI sites of pGEM11Zf(+). The vector was digested with BamHI and EcoRI to remove a 1.3-kb BamHI/EcoRI genomic fragment containing exon 10 and flanking intron sequences. The digested vector, without the 1.3-kb BamHI/EcoRI genomic fragment, was purified by gel purification and recircularized by ligation using three oligonucleotides: 5′-AATTGTCGACA-3′; 5′GATCGTCGACA-3′; and 5′-TCGATGTCGAC-3′. The recircularized vector, resulting from the ligation of the long arm to the short arm of homology, contained a unique SalI site where the 1.3-kb exon 10 region was deleted.