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 pS V2neo containing the neomycin-phosphotransferase gene was microinjecte d into the nuclei of NIH/3T3 cells synchronized by serum starvation an d aphidicolin treatment. The frequency of neo(r)-transformation (expre ssed as a percentage of microinjected cells) was 6% at the GO phase an d 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 decre ased in the parallel with the BrdU-labeling index. Furthermore we deve loped a simplified method in which asynchronously growing cells were t reated with aphidicolin at 10 mu g/ml for 16 hrs without serum starvat ion and subjected to microinjection, and their growth was further indu ced 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 ind icates that synchronized animal cells progressing from the G1/S bounda ry to the early S phase integrate the pSV2neo DNA into their chromosom es with high efficiency.