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: (I) a mechanism for making la rge numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select t ransgenic cells; and (3) a mechanism for site-specific gene transfer o r deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (I CM) cells, morulae and the precompaction 16-20-cell stage. All have ex hibited similar morphology to mouse embryonic stem (ES) cells, pluripo tency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or l ong-term cultures on bovine or murine fibroblast feeder layers, in eit her a microdrop or a culture dish. The relative merit of culture syste ms 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, totip otency was demonstrated by the birth of four carves from ICM cells cul tured 27 days or less in a loose suspension microdrop. Advanced plurip otency and perhaps totipotency was demonstrated in one fetus in a rece ntly reported study where morulae cells cultured in vitro were chimaer ized with non-cultured cells. DNA fingerprinting to associate cell lin es with offspring and karyotyping to ascertain chromatin normalcy is i mportant in ES cell research. Data pertaining to the use of each are p resented.