Serum starvation and full confluency for cell cycle synchronization of domestic cat (felis catus) foetal fibroblasts.
Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < or = 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < or = 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.
de Barros
F R O
FR
Laboratory of In Vitro Fertilization, Cloning and Animal Transgenesis, Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of Sao Paulo, Sao Paulo, Brazil. flaviarob@usp.br
Goissis
M D
MD
Caetano
H V A
HV
Paula-Lopes
F F
FF
Peres
M A
MA
Assumpção
M E O A
ME
Visintin
J A
JA
D000818:Animals; D002415:Cats / Q000196:embryology; D002453:Cell Cycle / Q000502:physiology*; D019976:Cloning, Organism / Q000662:veterinary; D016895:Culture Media, Serum-Free; D004247:DNA / Q000032:analysis; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000662:veterinary; D016193:G1 Phase; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D016192:Resting Phase, Cell Cycle
D016895:Culture Media, Serum-Free; D004247:DNA