PRODUCTION OF CALVES BY TRANSFER OF NUCLEI FROM CULTURED INNER CELL MASS CELLS

We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to prod uce totipotent blastocysts that resulted in calves born. Of 15 cell li nes represented in this study, 13 were derived from immunosurgically i solated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of att empted cell lines became established cell lines when started from 3 IC Ms. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plu s selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FC S) for 6-101 days before use in nuclear transfer, at which time some h ad multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nucle ar transfer clones were made by fusing the ES cells into enucleated oo cytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastoc ysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typin g showed the calves to be derived from the respective sires of the cel l lines. The calves were derived from cultures of less than 28 days.