Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.
We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.
Watanabe
M
M
Mammalian Development Laboratory, National Institute of Genetics, Mishima, Japan.
Shirayoshi
Y
Y
Koshimizu
U
U
Hashimoto
S
S
Yonehara
S
S
Eguchi
Y
Y
Tsujimoto
Y
Y
Nakatsuji
N
N
D017501:Adenovirus E1B Proteins / Q000235:genetics; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D002130:Calcium Phosphates; D002455:Cell Division / Q000235:genetics; D048429:Cell Size; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D018274:Electroporation; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D005854:Germ Cells / Q000166:cytology*; D008081:Liposomes; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection / Q000379:methods*; D051020:bcl-X Protein
D017501:Adenovirus E1B Proteins; D000952:Antigens, Polyomavirus Transforming; C494812:Bcl2l1 protein, mouse; D002130:Calcium Phosphates; D008081:Liposomes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C485828:alpha-tricalcium phosphate; D051020:bcl-X Protein; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous