THE CHROMOSOME MAKE-UP OF MOUSE EMBRYONIC STEM-CELLS IS PREDICTIVE OFSOMATIC AND GERM-CELL CHIMERISM

Mouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, incl uding the germline, of an organism referred to as a chimaeric. However , the reasons why this contribution often appears erratic are poorly u nderstood. We have tested the notion that the chromosome make-up may b e important in contributing both to somatic cell chimaerism and to ger m line transmission. We found that the percentage of chimaerism of ES cell-embryo chimaeras, the absolute number of chimaeras and the ratio of chimaeras to total pups born all correlate closely with the percent age of euploid metaphases in the ES cell clones injected into the muri ne blastocyst. The majority of the ES cell clones that we tested, whic h were obtained from different gene targeting knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline ; in contrast, none of the ES cell clones with more than 50% of chromo somally abnormal metaphases transmitted to the germline. Euploid ES ce ll clones cultured in vitro for more than 20 passages rapidly became s everely aneuploid, and again this correlated closely with the percenta ge of chimaerism and with the number of ES cell-embryo chimaeras obtai ned per number of blastocysts injected. At the same time, the ability of these clones to contribute to the germline was lost when the propor tion of euploid cells dropped below 50%. This study suggests that aneu ploidy, rather than 'loss of totipotency', in ES cells, is the major c ause of failure in obtaining contributions to all tissues of the adult chimaera, including the germline. Because euploidy is predictive of g ermline transmission, karyotype analysis is crucial and time/cost savi ng in any gene-targeting experiment.