Developmental competence of nuclear transfer cow oocytes after direct injection of fetal fibroblast nuclei.
We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.
Lacham-Kaplan
O
O
Center for Early Human Development, The Monash Institute of Reproduction and Development, 27-31 Wright Street, Clayton, Victoria 3168, Australia. Orly.Lacham-Kaplan@monash.edu.au
Diamente
M
M
Pushett
D
D
Lewis
I
I
Trounson
A
A
D000034:Abortion, Veterinary; D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002843:Chromatin / Q000648:ultrastructure; D019976:Cloning, Organism / Q000379:methods*; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D013045:Species Specificity; D013997:Time Factors
D002843:Chromatin; D007476:Ionophores; D002118:Calcium