Cell synchronization for the purposes of nuclear transfer in the bovine.
We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.
Korfiatis
N
N
Centre for Early Human Development, Monash Institute of Reproduction and Development, Clayton, Victoria, Australia. natasha.korfiatis@med.monash.edu.au
Trounson
A
A
Lacham-Kaplan
O
O
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle / Q000196:embryology / Q000235:genetics*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000662:veterinary*; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004622:Embryo, Mammalian / Q000502:physiology; D005314:Embryonic and Fetal Development; D005347:Fibroblasts / Q000187:drug effects / Q000648:ultrastructure; D007621:Karyotyping; D009028:Morula / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D010312:Parthenogenesis; D011003:Ploidies
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D015739:Nocodazole