Construction Of A Murine Hair-follicle-cell-Specific Expression Vector Of KGF And Screening And Identification Of Transgenic Embryonic Stem Cells

Keratinocyte growth factor (KGF) that stimulates the proliferation of keratinocyte and other epithelial cells is a member of the fibroblast growth factor family, also is named as FGF-7. It has been found that KGF is expressed in hair papilla cells of growth. It can specially bind with KGF receptor in hair follicle, then stimulate the growth of hair follicle, which explains that KGF has the function of supporting the growth of hair follicle. As important method of studying gene function, the construction of hair-follicle-cell-specific expression vector and production of transgenic animal model could be used to probe KGF gene's role and regulation mechanism during the development of mouse hair follicle. In order to interpret the function of KGF gene during the development process of mouse hair follicle, transgenic animal technology was used in this study. The hair-follicle-cell-specific expression vector pCDsR-UKA was introduced into mouse embryonic stem cells by Lipofectamine2000, then the transgenic ESCs were screened by G418 to obtain the ES cells of trans-ATGF gene and red fluorescin steadily expressed. The transgenic ESCs could be the nuclear donors to produce transgenic mouse for KGF overexpressed, moreover through constructing transgenic mouse model, this might provide research foundation for studying the role of KGF gene for mouse hair follicle.1. The construction of the hair-follicle-cell-specific expression vector pCDsR-UKA.The mouse keratinocyte growth factor (KGF) coding region cDNA sequence was amplified from mouse fibroblast total RNA by RT-PCR technology, then linked with T vector pBlue-T to obtain an intermediate vector pB-KGF. At next step, the UHS promoter and BGH polyA tail fragment were ligated between SacⅠ+XbaⅠand EcoRⅤ+SalⅠsites of pB-KGF vector respectively, then the UHS+KGF+polyA gene fragment was ligated between SacⅠ+SalⅠsites of pCDsRed2 expression vector to construct the hair-follicle-cell-specific expression vector pCDsR-UKA(6.6kb). The structure and sequence of expression vector pCDsR-UKA were consistent with expected design based on DNA sequencing results and restriction endonucleasa assay.2. Isolated culture of mouse embryonic fibroblasts and production of feeder layer in vitro.The mouse embryonic fibroblasts (MEF) were successfully isolated by monoplast inoculation method from a 13.5d's mouse fetus, then they were in vitro cultured and passaged in high-glucose DMEM+10%FBS medium at 37℃, 100% humidity and 5%CO2 incubator. The MEF from 1st to 3rd generation that were dealed with 20μg/mL MMC was made as feeder layer. Then the ESCs were passaged on feeder layer and in vitro cultured in 80% high-glucose DMEM+15%FBS+LIF ESM with other necessary factors at 37℃,100% humidity and 5%CO2 incubator. Meanwhile the growth curves of the 2nd MEF and SNL cell lines were drawed, chromosomal analysis of the ES cells was performed and ESCs were stained and analyzed by AKP. Besides, the Oct-4 and SSEA-1 immunofluorescence dye were also analyzed. The results above proved that the ESC clones in vitro cultured growed normally and were not differentiated. So they could be used for the research of transfection efficiency with liposome method.3. The condition optimization of exogenous gene transfection of mouse ES cells by liposome and screening and identification of transgenic ESCs.In this experiment, the toleration mensuration of ESCs on G418 was examined, the optimal screening concentration of G418 determined by experiment result of the concentration gradient dilution was 800μg/mL and sustainal concentration of G418 was 400μg/mL.When transfecting mouse ESCs by Lipofectamine2000, we made a gradient optimization on DNA concentration, liposome dosage and transfection incubation time and so on. After 48 hours, we gathered statistics on the transfection efficiency of different groups to obtain the best transfection condition. With optimized liposome suspension transfection method, we found that when the ratio of DNA and liposome was 3:10 in 6-well culture plate resulted in the highest transfection efficiency that was up to (34.4±4.1)%. The ESCs transfected after 48 hours had been screened by 800μg/mL G418 for 4-5 days. After sustainly screened by 400μg/mL G418 for 5-6 days in embryonic stem cell medium at 37℃incubator, transgenic ESC clones with red fluorescent were obtained. Identification of the genomic DNA of transgenic ESCs by PCR amplification proved that the exogenous KGF gene had steadily integrated into genomes of the ESCs. Meanwhile chromosomal analysis of the transgenic ESCs and identification of AKP, Oct-4, SSEA-1 immunofluorescence dye proved that the transgenic ESCs growed normally and did not differentiate in vitro. They could be donor cells for tetraplont blastula so that we can produce ES mouse model, which might make a great effort for studying KGF gene that can regulate and promote the mouse hair follicle in vitro.