[Knockout serum replacement improves establishment efficiency of C57BL/6J mouse embryonic stem cell line].
OBJECTIVE
To eliminate the influence of serum on self-renewal of embryonic stem cells (ESCs), knockout serum replacement (KSR), a defined formulation, was used to replace serum for the establishment of C57BL/6J mouse ESC line.


METHODS
C57BL/6J mouse blastocysts collected at 3.5 days post coitum (d.p.c.) were cultured in the medium supplemented with KSR. In control experiment, KSR was substituted by fetal bovine serum (FBS). When ESC line was established, the morphology of ESCs, the expression of alkaline phosphatase and oct-4, and the karyotype and differentiating ability of ESCs were analyzed.


RESULTS
13 blastocysts were cultured in the medium supplemented with KSR and one ESC line (MES-1) was established with a normal and stable XX karyotype after cultured for more than 20 passages, and then the high expression of alkaline phosphatase and oct-4 was detected. When cultured in suspension, MES-1 formed embryoid bodies. When inoculated subcutaneously into nude mice, MES-1 formed teratoma. After injected into ICR mouse blastocysts collected at 3.5 d.p.c., MES-1 incorporated into the inner cell mass of the host blastocyst and contributed to the development of a chimera. In control experiment, no ESC lines were cultured for more than 3 passages.


CONCLUSIONS
KSR can be efficiently used to isolate and culture C57BL/6J mouse ESCs, which can eliminate traditional prescreening of FBS suitable for isolation and culture of ESCs.
Wang
Hong
H
Institute of Biotechnology, School of Life Science and Technology, Tongji University, Shanghai, China.
Tian
Hai-Bin
HB
Chen
Juan
J
Sha
Hong-Ying
HY
Chen
Jian-Quan
JQ
Cheng
Guo-Xiang
GX
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001755:Blastocyst / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007621:Karyotyping; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D050814:Octamer Transcription Factor-3 / Q000378:metabolism
D003470:Culture Media; D050814:Octamer Transcription Factor-3; D000469:Alkaline Phosphatase