Tetracycline Egulatable Gene Expression Systems For Theexpression Of LEF1in Fibroblasts

Tetracycline regulatable gene expression systems is developed in recent years, having efficient and closed switch function. We can control gene expression in the transgenic cell and transgenic animals from the perspective of time and space,with the tetracycline regulatable gene expression systems.This study contrcted the epidermic-specific expression vectors for LEF1, with the tetracycline regulatable gene expression systems, and was transfected into goat fetal fibroblast cells with the vector PCDNA6. The tetracycline inducible expression of LEF1gene, detection of mRNA levels.1Construction of vector of pcDNA4-KL LEF1gene follicle-specific expressionThis study applicanted the tetracycline regulatable gene expression systems, the system of expression vector pcDNA4as the basic framework, through PCR amplification,got keratin associated protein6-1gene amplified (KAP6-1) promoter region sequence and sheep lymphoid growth factor I (LEF1) coding region sequences. Vector pcDNA4was deleted sequences by restriction enzyme,PCR and ligase. The promoter of KAP6.1gene replaced the original promoter CMV, sheep lymphoid growth factor I (LEF1) was inserted into the multiplecloning sit. Through restriction endonuclease analysis for partial sequence,the result showed that the constructed expression vector pcDNA4-KL matched the structure of sequences with the expected design in this experiment. Lay the foundation for expression system of vector pcDNA6transfected cells for the next step and a tetracycline-regulated.2Cashmere goat fetal fibroblast cells isolated, cultured in vitro and exogenous gene transfectionCultivate fetal skin fibroblast in vitro, and carry on the double vevtor transfection cell experiment. Configurate DMEM/F12cell culture medium containing10%FBS, cultivate at37℃and5%CO2, saturate humidity conditions in vivo. When cell growth covered the whole dish, cryopreservation of cells. We changed cell culture medium once two days, when cell growth covered the entire cell cultures, cryopreservated the cells in cell cryopreservation solution, that was10%DMSO+90%FBS. By liposome mediated method, the expression vector pcDNA4-KL and expression vector pcDNA6were transfected into goat fetal fibroblast cells for second generations, and monoclonal cells were got by blasticidin and zeocin. monoclonal cells were digested by0.25%trypsin and expand trained.When cells were full of the whole dish, we extracted of genomic DNA, PCR identification. Concentration of blasticidin for13μg/ml and the zeocin concentration is1500μg/ml, two common antibiotics screened12days, we obtained monoclonal cells. Cloning cup of trypsin digestion method, expand the trained monoclonal cells, and cell genomic DNA were extracted with common gene DNA extraction kit, PCR amplification and electrophoresis appraisaled expression vector pcDNA4-KL had integrated with the cell genome stability, and the expression vector pcDNA6and cell genome also integrated stability. Inducing of the next step of tetracycline and its detection were proceed.3Tetracycline inducible gene expression of LEF1in fibroblastsThe transgenic fibroblasts at different concentrations (Oug/ml lug/ml lOug/ml20ug/ml) were induced by tetracycline, which has hair follicle specific expression vector pcDNA-KL and inducible expression vector pcDNA6transgenic, were cultured in the10%FBS+DMEM/F1237℃and5%CO2, induced saturated humidity conditions to develop. After24h, extract RNA under different tetracycline induced cells. Through the reverse transcribed into cDNA, real-time method to detect lymphoid enhancement factor (LEF1) on the expression of mRNA level, determine the optimal concentration of tetracycline.