Northwestern University Feinberg School of Medicine

Center for Genetic Medicine

Mating TTML Animals

General mouse mating schemes

Separate (singly house) males at seven weeks of age to optimize sperm counts. Introduce two females (over six weeks old) to each singly housed male the following week. Check for the presence of a copulation plug every day for a week. Remove the first two females after seven days and add two fresh females to the cage. If plugs were observed, expect litters 19 days from day of plug. Note that males, particularly young males, will sometimes mate with females that are not in estrus so the females may not be pregnant or may drop a litter several days later than expected, if the male mates with her again when she is in estrus (the female estrous cycle occurs every four to five days). Plugs may be observed even if the male is sterile; in which case, no litters will ever be born.

As an alternative to checking for plugs, weigh females just prior to mating. Check weight in 10 days. A weight gain of five or more grams in C57BL/6 females is usually indicative of a pregnancy. If unsure, weigh females again in two-day intervals. Any weight gain at this point generally denotes pregnancy.

Mating schemes for 129-derived chimeras

Remember that chimeric animals are composed of tissues, including germ cells, derived from both the ES cell and the blastocyst. Therefore, 129 ES cell-derived chimeric mice mated back to the same wild-type C57BL/6 strain used to generate the blastocysts for injection will produce both black and agouti offspring (Mouse Strain Coat Colors). Black pups have inherited two black coat color alleles (a/a; C/C), one allele from the wild-type mate and the second from the ICM cells of the C57BL/6 blastocyst, and are of no further interest. Agouti pups (a/ Aw) have inherited one black coat color allele from the wild-type mate and the agouti allele, Aw, from the ES cells. Agouti pups, like the black pups, will have black skin and it will take about seven to 10 days until the hair grows in to determine germline transmission of the ES genes, so be patient. Agouti hair on these mice has a yellow band on an otherwise black strand. Agouti coloration by itself signifies the germline transmission of ES cell genes (i.e., that the ES cells which incorporated into the ICM of the blastocyst following microinjection are/were capable of giving rise to germ cells) but is not indicative of the inheritance of the mutated allele and therefore, all agouti pups must be genotyped for the presence of the mutation.

Mating schemes for B6-derived chimeras

C57BL/6 (a/a; C/C) ES cell line microinjected into blastocysts from the albino B6(Cg)-Tyrc-2J/J line create black and white chimeric animals. Chimeras are then mated back to wild-type albino B6(Cg)-Tyrc-2J/J mice. Germline transmission of ES cell derived genes can be identified at birth by the presence of pups with black eyes, due to the inheritance of the dominant C allele at the albino locus from the C57BL/6 ES cell. These pups will be black in color and must be genotyped for the inheritance of the mutated allele.