Molecular Note
A serine 123 to alanine (S123A) substitution was performed by mutating the TCT codon in exon 5 to GCT. A loxP-flanked neomycin cassette was also included upstream of exon 5 for targeting purposes. Mice generated by correctly targeted ES cells were crossed with cre-transgenic mice to remove the neo cassette leaving behind a single loxP site. Sequence analysis of cDNAs from homozygote embryonic fibroblasts demonstrated the mutated allele was expressed. Immunoblot analysis indicated no differences in basal expression between mutant and wild-type proteins. The mutant protein was found to have a 50% longer half-life than wild-type protein in embryonic fibroblasts.
