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 microinjecting a nucleus directly into the cytoplasm of the oocyte. A major mammalian 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). Small, confluent and serum starved cells resulted in nuclear formation more often (P < 0.048) than did cycling cells. The rate of nuclear formation was not 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 cells were used for nuclear transfer and resulted in two presumptive blastocyst stage embryos [2/128 injected or 2/38 (5.3%) successful injections]. These results show that presumptive blastocyst stage embryos can result from microinjection of fibroblast cells to enucleated oocytes and thus may provide a method to create transgenic knockout animals.
Tao
T
T
Department of Animal Sciences, University of Missouri-Columbia, 65211, USA.
Machàty
Z
Z
Boquest
A C
AC
Day
B N
BN
Prather
R S
RS
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D002843:Chromatin / Q000737:chemistry; D005260:Female; D005347:Fibroblasts / Q000502:physiology*; D008297:Male; D008845:Microinjections / Q000662:veterinary*; D009865:Oocytes / Q000502:physiology*; D013552:Swine / Q000196:embryology*
D002843:Chromatin