Synchronization of goat fibroblast cells at quiescent stage and determination of their transition from G0 to G1 by detection of cyclin D1 mRNA.
A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G0), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G1 phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G0 or G1 cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G0 to G1 by detecting G1-specific marker, cyclin D1 mRNA. Fluorescent-activated cell sorting (FACS) analyses of cells after 4 days of serum starvation showed that more that 90% of cells were in G0/G1. Additionally, detection of cyclin D1 mRNA by northern blot analysis showed that 4-day serum starved quiescent cells started entering G1 a few hours after addition of 10% serum to the medium. Taken together, the data indicated that serum starved transfected primary fibroblasts of adult goats experienced the G0 to G1 transition within 5 h of serum stimulation and were at the mid-G1 stage within 10 h of serum stimulation.
Memili
Erdogan
E
GTC-Biotherapeutics Inc., Framingham, Massachusetts 01701, USA.
Behboodi
Esmail
E
Overton
Susan A
SA
Kenney
Anna M
AM
O'Coin
Michael
M
Zahedi
Arya
A
Rowitch
David H
DH
Echelard
Yann
Y
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase / Q000502:physiology*; D006041:Goats; D012333:RNA, Messenger / Q000378:metabolism*; D016192:Resting Phase, Cell Cycle / Q000502:physiology*
D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D019938:Cyclin D1