Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes

Nuclear transfer as originally developed for use in amphibians involved mic roinjecting a nucleus directly into the cytoplasm of the oocyte. A major ma mmalian modification has been to use cell fusion to introduce the nucleus. Here we report using a microinjection method to introduce small and medium sized fibroblast cells into mature oocytes. Small cells were more likely to result in nuclear formation (30%) than larger cells (15%; P = 0.013). Smal l, confluent and serum starved cells resulted in nuclear formation more oft en (P < 0.048) than did cycling cells. The rate of nuclear formation was no t dependent upon the media, (NCSU-23 or TL-Hepes without calcium) nor upon the duration of exposure to the media (1 h to 4 h) after microinjection but before activation. While such treatments did not have an effect on nuclear formation, treatment of parthenogenetically activated oocytes with calcium -free TL-Hepes reduced the percentage of blastocysts (P = 0.068; 11.2% vs. 18.3%) and increased the percentage of morula stage embryos (P = 0.007; 27. 6% vs. 15.7%) as compared with culture in NCSU. Finally, small confluent ce lls were used for nuclear transfer and resulted in two presumptive blastocy st stage embryos [2/128 injected or 2/38 (5.3%) successful injections]. The se results show that presumptive blastocyst stage embryos can result from m icroinjection of fibroblast cells to enucleated oocytes and thus may provid e a method to create transgenic knockout animals. (C) 1999 Elsevier Science B.V. All rights reserved.