Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells.
Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 +/- 6.0 cells; serum-starved group: 86.8 +/- 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.
Gibbons
John
J
Department of Animal and Dairy Sciences, University of Georgia, Athens, GA 30602, USA.
Arat
Sezen
S
Rzucidlo
Jacek
J
Miyoshi
Kazuchika
K
Waltenburg
Rachel
R
Respess
Donald
D
Venable
Alison
A
Stice
Steve
S
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D042846:CDC2-CDC28 Kinases; D002417:Cattle / Q000235:genetics / Q000502:physiology*; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D019976:Cloning, Organism; D016895:Culture Media, Serum-Free; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004247:DNA / Q000032:analysis; D004624:Embryo Transfer; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000648:ultrastructure; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D011256:Pregnancy Outcome; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine
D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D011687:Purines; D000077546:Roscovitine; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases