Generation of mice with targeted disruption of the sam68 gene.
A λ bacteriophage clone encompassing Sam68 exons 3 to 9 was isolated from a 129/SvJ genomic library using full-length Sam68 cDNA as a probe. XbaI-digested Sam68 genomic DNA fragments of 4 kb (encompassing exon 4 and part of exon 5) and 3kb (spanning part of exon 5 and exon 6) were subcloned in Bluescript SK resulting in pBS4 and pBS3, respectively. A DNA fragment was amplified from pBS4 with the following oligonucleotides (5′-AAT GTC TAG AAA CAA CTC ATA TAC AGA C-3′) and the universal primer. The XbaI-digested 1-kb DNA fragment was subcloned in the XbaI site of pPNT (a gift from Andrew Karaplis, McGill University, Montréal, Quebec, Canada). The 3-kb fragment from pBS3 was amplified by PCR with (5′-GGG ATG CGG CCG CTC TAG AAT TGT CCT ACT TGA ACG G-3') and (5′-CGG TGG CGG CCG CTG TCG ACC TGA GTA ACA TTT CTT A-3′) and subcloned in the NotI site of pPNT. The targeting vector pPNT-Sam68 replaces exon 4 and part of exon 5 with a neomycin-resistant gene cassette. An SalI site was introduced at the 3′ end of the 3-kb DNA fragment and was used to linearize the plasmid for electroporation into embryonic stem (ES) cells. Approximately 1,000 ES colonies were screened and two clones were identified that contained the Sam68 mutant allele, as determined by Southern blotting. Targeted ES cells were injected into 3.5-day-old BALB/c blastocysts and were transferred into CD-1 foster mothers, and animals classified as chimeras by coat color were mated with BALB/c mice. Germ line transmission was achieved and the mice were maintained in C57BL/6 background. The mice used for this study represent mice that were backcrossed in C57BL/6 between three and eight generations; in addition, we maintained the mice in the 129/SvJ strain and observed a similar phenotype.