Gene Targeting to Generate Mlh3-D1185N Mice

A PL253 targeting vector containing the Mlh3-D1185N point mutation in the potential endonuclease domain and a loxP-neo-loxP cassette in intron 5–6 of Mlh3 was incorporated into an embryonic stem cell line. Mlh3^DN transgenic mice were crossed with a Spo11-Cre mouse line to remove the neo cassette [76 (link)], and then maintained on an inbred background through backcrossing on to the C57Bl/6J line (Jackson Laboratory, Bar Harbor, ME). Genotyping of WT, Mlh3^+/DN, and Mlh3^DN/DN mice was performed using the following PCR primer pairs: forward (5’-AAGCCAAGTCTGCATGAGTA-3’) and reverse (5’-TAAATGTGCCACTGACTAAAT-3’) followed by a restriction enzyme digestion with Sau96I (New England Biolabs) at 37°C for 2–3 hours, which results in 439-bp and 263-bp fragments from the WT allele and a 702-bp fragment from the mutant allele. Fertility tests were performed by breeding Mlh3^DN/DN adult males with WT females. At least 3 males of each genotyped were evaluated. Presence of a copulation plug the following morning counted as a successful mating event. Pregnancy was confirmed by gentle palpation of the abdomen after gestation day 11 or on delivery date of litters. Mus81^-/- animals were generated from our breeding stock of such mice, as previously described [6 (link)]. Mice were housed and utilized under the guidance and approval of the Cornell University Institutional Animal Care and Use Committee.