Map2k1^tm2.1Chrn

The gene was targeted to reproduce the most common human mutation associated with cardio-facial-cutaneous (CFC) syndrome by creating an A-to-G transition in exon 3 at the base corresponding to nucleotide position 389 of the cDNA, resulting in replacement of tyrosine by cysteine at amino acid position 130 of the protein (Y130C). A loxP-flanked neomycin selection cassette was introduced and subsequently deleted by Cre recombinase, leaving a single loxP site in intron 2. Sequence analysis revealed a 61.4-kb intragenic duplication beginning 5.6 kb upstream of the transcription initiation site and extending into the fifth intron, with a single break junction between intron 5 of the 5' copy and the upstream sequence of the 3' copy suggesting it arose through unequal crossing over. Southern blot analysis using a restriction endonuclease that cuts within the intron 2 loxP sequence showed that the Y130C point mutation resides in the 5' copy. Total Map2k1 mRNA abundance in kidneys, lungs and thymi of mice heterozygous for this and a null mutation (Map2k1tm1Chrn) was shown via qRT-PCR to be about half that in wild-type (Map2k1+/+) tissues; sequence analysis of the qRT-PCR products revealed the presence of both Y130C mutant and wild-type transcripts in the compound heterozygotes. Similarly, immunoblot analyses demonstrated total MAP2K1 protein levels in both Map2k1tm2.1Chrn/ Map2k1tm1Chrn (Mek1Y130C/-) and Map2k1+/ Map2k1tm1Chrn (Mek1+/-) tissues ~50% of wild-type (Mek+/+) levels. (J:261678)