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A targeting vector was constructed to engineer a point mutation into the gene locus. A P394L mutation (a C-to-T transition) was introduced into exon 8 by PCR-based site-directed mutagenesis. This mutation is equivalent to the P392L substitution in the human gene. A loxP-flanked neo cassette was also introduced immediately downstream of exon 7. After targeting, transient cre-recombinase expression removed the neo cassette leaving a single loxP site behind. Southern blot and genomic PCR analysis confirmed correct targeting of the construct. (J:141179)