The Establishment of Both G418 and Hygromycin B Resistant NIH3T3 Cell Line as Feeder Layer for Mouse ES Cell Culture

Abstract: In order to establish a both G418 and hygomycin B resistant NIH3T3 cell line as the feeder layer for ES cell culture in pTet-on system， the two plasmids pWL/neo containing neo gene and pHyg containing hygromycin gene were transfected by one after another into NIH3T3 cells with lipofectin method. For each plasmid transfection the transfected cells were subjected to antibiotics selection， under the 500 mg/ml G418 and 300 mg/ml hygromycin B selection several both resistant NIH3T3 cell clones have been successfully selected. In further examination the both G418 and hygromycin B resistant NIH3T3 cells can grow normally in selection medium and these stable transfected cell clone can be passed by generation and generation， they were no different from normal NIH3T3 cells in morphology. Meanwhile the PCR method were performed to check the integration of the foreign DNA fragments， when the primers for the neo gene and hygromycin gene were applied in these PCR reactions， the specific neo gene fragment and hygromycin gene fragment were amplified in G418 and hygromycin B resistant NIH3T3 cell DNA. Furthermore the feeder layer prepared from these both resistant NIH3T3 cells was used for mouse ES cells culture， we observed that these ES cells grew very well either in morphology or growth situation as same as normal NIH3T3 cell feeder layer. The establishment of these both G418 and hygromycin B resistant NIH3T3 cell clones provided powerful tool in mouse ES cell culture， when the ES cells will be selected by drugs after pTet-on and pTRE-H-Insulin transfection in tetracyclin inducible expression system.

CSTR: 32200.14.cjcb.2005.02.0023