Abstract: Objective In order to obtain a feeder layer of ES cells transfected by pTRE-Ins-human gene by means of the hygromycin-resistant NIH3T3 cell line established.Methods The plasmid pHyg containing hygromycin phosphofransferose gene was purified and transfected into NIH3T3 cells with Lipofectin.The transfected cells would be survived in further culture medium containing hygromycin B antibiotic as the hygromycin phosphofransferose gene could express a hygromycin B resistant products.The identification of the hygromycin B resistant NIH3T3 cell line was conducted by polymerase chain reaction with the specific primers of hygromycin phosphofransferose gene. Results Under the condition of hygromycin B（300μg/ml ），hygromycin B resistant NIH3T3 cell clones have been successfully selected.The hygromycin B resistant NIHNIH3T3 cells were no differences from normal NIH3T3 cells in morphology and propagation rate.The hyg phosphofransferose gene DNA fragment could be amplified by PCR with specific primers in the genomic DNA of hygromycin B resistant NIH3T3 cells；Southern blot analysis showed that hygromycin phosphofransferose fragment was integrated into NIHNIH3T3 cells. Conclusion The hygromycin B resistant NIH3T3 cells were established successfully by Lipfectin,which may be useful of selecting ES cells transfected by pTRE-Ins-human gene.

Key words: NIH3T3 Cell, Hygromycin B phosphofransferose gene, Transfection, Hygromycin B resistant, ES Cell