|
KMID : 0894520080120010077
|
|
Development & Reproduction
2008 Volume.12 No. 1 p.77 ~ p.86
|
|
|
Derivation of Mouse ES Cells from Isolated Blastomeres in Culture Media Supplemented with LIF
|
|
Cho Jae-Won
Lim Chun-Gyu
Ko Duck-Sung
Kang Hee-Jung
Jeon Jin-Hyun
|
|
|
Abstract
|
|
|
|
This study was carried out to investigate the effect of leukemia inhibitory factor (LIF) on the derivation of mouse ES cells from isolated blastomeres. Two-cell stage mouse embryos were obtained from superovulated BDFl female mice. Collected embryos were cultured to blastocyst stage in culture medium supplemented with 0, 1,000, 2,500 or 5,000 U/mL of LIF. Cultured blastocysts were examined by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE) using differential staining method. When 2-cell embryos were cultured with 2,500 U/ml of LIF, the cell numbers of ICM significantly increased in comparing with those of the control (21.0±4.0 vs. 15.9±5.0, P<0.01) and 1,000 U/mL of LIF-containing group (21.0±4.0 vs. 16.6±4.9, P<0.05). We used an ES cell establishment medium with 20% Knockout Serum Replacement and 0.01 mg/mL ACTH instead of fetal bovine serum. Establishing efficacy of ES cell lines were the highest in 2,500 U/mL of LIF-containing group as 36.7% (11/30). This culture medium was applied to the culture of isolated blastomeres and to derivate ES cell lines. Three ES cell lines (21.4%) from isolated blastomeres of 2-cell stage embryos were established. In further experiments, we could establish one ES cell line (4.0%) from single blastomere of 4-cell stage embryo. The subcultured ES cells and their embryoid bodies were characterized by analyzing gene expression for undifferentiation and differentiation marker gene using immunocytochemistry and RT-PCR. In conclusion, LIF supplementation in culture medium could increase the cell number in ICM of blastocysts and support derivation of ES cell lines from isolated blastomeres.
|
|
|
KEYWORD
|
|
|
LIF, ICM, Mouse ES cells, Isolated blastomere, Gene expressions
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|
|