Preparation of a G418-resistant feeder layer of NIH3T3 cells
- VernacularTitle:具G418抗性NIH3T3细胞饲养层的制备
- Author:
Meiying ZHANG
;
Hua LI
;
Wanwei DONG
;
Wei YANG
;
Ying WANG
;
Ying QIN
;
Luzeng WANG
;
Taiyi WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(37):144-147,封三
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Establishment of drug (neomycin or hygromycin) resistant feeder layer cells is necessary for screening the target gene positive clone of embryonic stem cell (ESC) transfected in vitro, and the establishment of drug-resistant NIH3T3 cell line is also important for the screening of other target ESC genes.OBJECTIVE: To obtain a feeder layer of positive cell clone of ESC transfected by pTet-on gene by means of the G418-resistant NIH3T3 cell line establishment.DESIGN: Cell culture and DNA examination.SETTING: Faculty of Laboratory Animal, China Medical University.MATERIALS: NIH3T3 cells were contributed by the Cell Biology Staff Room of China Medical University, and pWL/neo plasmid was a gift from Professor Jin Zhuang of Harvard University Medical College. G418,leukemia inhibitory factor (LIF, ESC GRO 106 U/mL) and DMEM were produced by GIBCO BRL Company, while mitomycin-C (MIT-C), DIG marking and kits were the products of Roche Company. Lipoetin was the product of Invitrogen Company, and bought from Shenyang Lianxing Biotechnology Co.,Ltd.METHODS: ①The pWL/neo plasmid containing neo gene was purified and transfected into NIH3T3 cells by lipoetin.②The transfected cells were further cultured in 25 mL medium containing G418 antibiotic of gradient concentration to observe the survival and apoptosis of cells and measure G418 minimal fatal dose (MFD) to NIH3T3 cells.③The transfected cells were subcultured, and the clone of single cell was selected to 24-pore culture board for screening amplification. At the same time, normal NIH3T3 cells were also selected and added with the selective culture medium at the same dose of G418, as screening negative control. ④The cells were planked with lower cell density, and further screened in DMEM medium containing G418 MFD; Meanwhile, normal NIH3T3 cells were taken as controls, and cultured by G418 contain MFD and by DMEM medium without G418 respectively. Light microscope was used to observe G418R NIH3T3 cells. ⑤G418R NIH3T3 cells and MEF were all applied as the feeder layer to culture the ES-D3 cells, which were observed by light microscope. Cell DNA was prepared, and evaluated by PCR and southern blot.MAIN OUTCOME MEASURES: ①G418 MFD to NIH3T3 cells. ②screening result of G418R NIH3T3 cells. ③growth of G418R NIH3T3 cells. ④growth of ES-D3 cells on G418R NIH3T3 cells.RESULTS: ①G418 MFD to NIH3T3 cells was defined as 500 mg/L.②Under the condition of G418 (500 mg/L), G418R NIH3T3 cell clones were successfully selected. ③The G418R NIH3T3 cells had no difference from normal NIH3T3 cells in morphology and propagation rate.④The ESC cultured in feeder layer present a colony growth, smooth limitation and remained an undifferentiation state. The resistant cell genomic DNA of G418R NIH3T3 cells was assayed with specific neo gene primer, and then neo gene DNA fragment was amplified; Southern blot analysis showed that neo gene fragment integrated into G418R NIH3T3 cells.CONCLUSION: The G418R NIH3T3 cells are established successfully,and the ESC cultured in the G418R NIH3T3 cell feeder layer can keep the characteristics of normal ESC.
