Question

You have isolated C57BL6 trophoblast stem cells and have propagated them on MEFs with heparin. To test for the success of your culture protocol, you are injecting the cells into a C57BL6 blastocyst. Once the pups are born, you notice that your experiment failed. Why? What was the expected outcome of this injection?

Answer 1

The targeted C57BL/6 ES cells are injected into a Balb/c recipient blastocyst (white coat color). The white Balb/c cells actually contain a masked agouti pigment, which is uncovered when it contacts the black pigment. Therefore, chimeras born from this mix of cells have patches of black and patches of white, with agouti hairs at the borders. Small patches of cells from the C57BL/6 ES cells will simply appear agouti on the white coat. To begin your new mouse colony, you can maintain your mutation on the C57BL/6 background while taking advantage of the fact that black is dominant to the white/masked agouti of Balb/c, and mate your C57BL/6-Balb/c mix chimeras to C57BL/6 mice. Progeny will be either black when the C57BL/6 ES cells have contributed to the chimera's germline, or progeny will be agouti(brown) when cells from the Balb/c recipient blastocyst have contributed to the germline. Again, Balb/c crossed to C57BL/6 will unmask the agouti gene of Balb/c, giving brown pups, which are not the pups you want this time. In this mating, all the black pups should be genotyped to find out which ones have received the targeted allele. Once again, most gene targetings only mutate one of the two alleles in the ES cell, so only 50% of the black mice will inherit a mutated allele. Mice with the targeted allele can be mated to each other or to wild type C57BL/6 mice and the background of the strain will remain pure C57BL/6.