Generation of Chaf1a mutant mice and embryos.
Using PCR, we amplified two genomic fragments (about 3 kb each) flanking Chaf1a exon 3. These DNA fragments were assembled by conventional cloning with the neomycin and DT (diphtheria toxin) cassettes, as described in Figure 1A. The construct was transfected in ES cells by electroporation. We identified recombinant ES cells carrying the mutant Chaf1a tm1Ger (abbreviated Chaf1a − in the manuscript) allele by Southern blot (Figure 1A). We derived Chaf1a +/− mice by injecting recombined ES cells into C57BL/6N blastocysts. E4 embryos obtained from the intercross of Chaf1a +/− mice were genotyped by nested-PCR amplification. Embryos were collected in a PCR reaction mix containing oligonucleotide primers 1, 2, and 3 (Figure 1A) that amplify the wild-type and mutant alleles. After 30 cycles of amplification, 1 μl of each PCR reaction was used in a second round of PCR amplification with a new set of oligonucleotide primers. After 30 cycles of amplification, PCR reactions were run onto an agarose gel, which revealed the presence of the wild-type (150 bp) and recombinant (200 bp) alleles.