An efficient transfection method for mouse embryonic stem cells.
Embryonic stem (ES) cells are considered to have potentials for tissue regeneration and treatment of diverse human diseases. ES cells are capable of indefinite renewal and proliferation, which can be induced to differentiate into tissues of all three germ lines. Despite these exciting potential, it remains unclear as to how the renewal and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockdown with small interfering RNA (siRNA) is commonly used in most of cancer or transformed cells but relatively rare in ES cells. In this study, we compare the transfection efficacies of several liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into mouse ES (mES) cells. Our results show that transfection by Effectene achieves the efficiency of >98% in CCE and >80% in D3 cells. The optimal ratio of DNA:Effectene for EGFP transfection is between 1:4 and 1:8. Transient-expressed EGFP or endogenous protein kinase A (PKA) were significantly knocked down by Effectene transfection of specific siRNA. High EGFP level expression and accumulation in mES cells induces minor cytotoxicity but can be reduced by introducing siRNA of EGFP. Further, this transfection method did not significantly affect mES properties of proliferation or differentiation. Our results provide an optimal protocol to achieve an efficient transfection for mES cells.
Ko
B S
BS
National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Chang
T C
TC
Shyue
S K
SK
Chen
Y C
YC
Liou
J Y
JY
D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D008055:Lipids / Q000008:administration & dosage; D008081:Liposomes; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage; D014162:Transfection / Q000379:methods*
C498175:Effectene; D008055:Lipids; D008081:Liposomes; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases