Cloning sheep from cultured embryonic cells.
The production of transgenic farm animals will be greatly enhanced with the development of cultured cell lines that remain totipotent following nuclear transfer. Here, data are presented that demonstrate the generation of both male and female cloned lambs from two established embryonic cell lines. Cytoplasts derived from in vivo oocytes resulted in slightly greater development to blastocyst (24% v. 17%) and survival to term (7% v. 2%) compared with in vitro oocytes. There was no advantage in co-culturing cloned embryos with oviductal epithelial cells compared with synthetic oviductal fluid medium in terms of development to blastocyst (18% v. 31%) or survival to term (both 8%). Although the survival of cloned embryos immediately after transfer was high based on 'biochemical' pregnancy, 64-80% of embryos failed over the attachment phase with in vivo cytoplasts. Although the co-transfer of trophoblastic vesicles improved embryo survival to Day 35 (45% v. 25%), there was no difference at term. A high proportion of fetuses were lost during the last trimester (43%), resulting in 11% of embryos transferred developing to term using in vivo cytoplasts (12/112). Five lambs have survived and two rams are fertile. The current nuclear transfer process is inefficient and further research is needed to improve the development of healthy fetuses.
Wells
D N
DN
Reproductive Technologies Group, AgResearch Ruakura, Hamilton, New Zealand. wellsd@agresearch.cri.nz
Misica
P M
PM
Day
A M
AM
Peterson
A J
AJ
Tervit
H R
HR
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000166:cytology; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D018920:Coculture Techniques; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology*; D005314:Embryonic and Fetal Development; D005187:Fallopian Tubes / Q000166:cytology; D005260:Female; D008297:Male; D018895:Microsatellite Repeats; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D011247:Pregnancy; D012756:Sheep / Q000235:genetics*