Efficient and stable gene transfer following microinjection into nuclei of synchronized animal cells progressing from G1/S boundary to early S phase.
We examined the possible phase(s) of the cell cycle in which a foreign gene can be stably transferred to animal cells. DNA of the plasmid pSV2neo containing the neomycin-phosphotransferase gene was microinjected into the nuclei of NIH/3T3 cells synchronized by serum starvation and aphidicolin treatment. The frequency of neo(r)-transformation (expressed as a percentage of microinjected cells) was 6% at the G0 phase and increased with progression of the cell cycle to reach a peak of 76% at the G1/S boundary. When the cells started their growth from the G1/S following release from aphidicolin, the frequency increased or decreased in the parallel with the BrdU-labeling index. Furthermore we developed a simplified method in which asynchronously growing cells were treated with aphidicolin at 10 micrograms/ml fro 16 hrs without serum starvation and subjected to microinjection, and their growth was further induced in aphidicolin-free medium. Using five cell lines (BALB/3T3, BALB/MK-2, NRK, CHO-K1, and HeLa) and one primary culture of chicken embryo fibroblasts (CEF), a 3- to 7-fold increase in the frequency of neo(r)-transformation was consistently detected in aphidicolin-treated cells, compared to non-treated asynchronous cultures. The present study indicates that synchronized animal cells progressing from the G1/S boundary to the early S phase integrate the PSV2neo DNA into their chromosomes with high efficiency.
Chida
K
K
Department of Cancer Cell Research, University of Tokyo, Japan. chida@ims.u-tokyo.ac.jp
Sueyoshi
R
R
Kuroki
T
T
D016475:3T3 Cells; D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D002642:Chick Embryo; D006224:Cricetinae; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics*; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D019868:Kanamycin Kinase / Q000235:genetics; D051379:Mice; D008845:Microinjections; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D051381:Rats; D016196:S Phase / Q000235:genetics*; D014170:Transformation, Genetic
D016590:Aphidicolin; D019868:Kanamycin Kinase