Systems for production of calves from cultured bovine embryonic cells

The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.