Abstract: 【Objective】To optimize the conditions for transfecting pluripotent transcription factors oct4、sox2、klf4 and c-myc mRNA to GEF cells, this study focused on transfection reagents and transfection methods. 【Method】GEF cells were separated from goat fetus leg muscle tissue via trypsin digestion method, and transcription factors then purified Using plasmid mini kits obtained from bacteriums. Then each downstream of the target genes in transcription carrier was extracted with a mono-restriction endonuclease enzyme. DNA purification kits were used to purify all the linear plasmids. Based on the linear plasmid, 5' end cap structure of mRNA was synthesised. TURBO DNase was used to eliminate the DNA template and the mMESSAGE mMACHINE® T7 Ultra Kit was use to add "tail" to the mRNA. Finally, MEGAclear™ Kit was used to purify the full mRNA. Five sheep derived recombinant plasmids: pCDNA3-oct4、pCDNA3-sox2、pCDNA3-klf4、pCDNA3-c-myc and pCDNA3-EGFP were linearized and then transcribed into mRNA in vitro respectively. GEF cells were transfected with different proportions of total mRNA (each of the five transcription factors mRNA was 0.2g). We used Lipofectamine 2000 by ratios of 1:0.5、1:1.0、1:1.5 and 1:2.0 to get the best transfection proportion. Using the best transfection proportions, transfection efficiencies were compared within the 2, 4, 6 generations of GEF cells. Immunofluorescence assay was performed to verify the transfection effect. 【Result】 The best transfection proportion was 1:1.0 (total mRNA/ Lipofectamine 2000), and the transfection efficiency of 2 generation was significantly higher than other generations of GEF cells under the best transfection proportion（P＜0.05）.This group of cells was good in both morphology and cell growth. Immunofluorescence assays showed that four pluripotent transcription factors mRNA were located in the nucleus of GEF cells, and obtained stable expression, while there was no expression of these four pluripotent transcription factors in the cytoplasm of GEF cells. After 24h of transfection by 4 pluripotent transcription factors, GEF cells turned slowly from fusiform to circular, and the cell activity was lower than the control group (normal GEF cells). 【Conclusion】 The best transfection condition for transfecting pluripotent transcription factors mRNA to GEF cells was at the 2 generation of GEF cells and under the 1:1.0 transfection proportion of total mRNA/ Lipofectamine 2000. This study provides proper parameters for transfecting cells or ES by mRNA , and also for obtaining iPS cells. We find indirect evidence for exogenous pluripotent mRNA in GEF cells and lay the foundation for further study of pluripotent transcription factors oct4、sox2、klf4 and c-myc.

Key words: mRNA, transfection, GEF, transcription factor