The Construction Of8S Hair Follicles Specific Expression Vector And Its Transgene Research

Spider silk has the superior characteristics of light weight, mechanical strength and toughness. Due to its exploitation difficulty, with the aid of bioengineering technologies to generate spider silk, becomes preferential strategy.8S spider silk protein gene is an octamer of core sequence S of the synthetic spider dragline silk protein gene. In order to further explore the functions and features of8S, in the study, an eukaryotic expression vector of specifically expressed8S gene in hair follicle cells were constructed. Then the expression vector was transfected into mouse epidermal cells and cashmere goat fetal fibroblast cells respectively, Then the activity of the tissue-specific promoter has been verified. Finally, mouse embryonic stem cells positive clones have been obtained by transfection, lay the foundation for the preparation of chimeric mice; simultaneously the8S transgenic mice were prepared by pronuclear microinjection technology, Our research lays a foundation for the further study of effects of8S on hair characteristics. 1. The construction of8S hair follicle specific expression vector pCDsR-K8SPPolyA fragment was amplified by PCR method, then linked with the vector pMD19T-8S to get an intermediate construction pMD19T-8S-PolyA. The hair follicle cells specific expression vector pCDsR-KAP6-1-IGF1and intermediate cloning vector pMD19T-8S-PolyA were digested by Kpn I/Sal I respectively, then pCDsR-K8SP was constructed by recombination. The evidence from restriction analysis and DNA sequencing showed that the vector was consistent with anticipation.2.8S hair follicle specific expression vector transfected cells and the identification of transgenic cellsThe hair follicle specific expression vector pCDsR-K8SP was transfected into mouse epidermal cells and cashmere goat fetal fibroblasts respectively by liposomes, and positive clones were obtained by double screening. Identification the genome of transgenic cells by PCR showed exogenous gene8S has been stably integrated into the genome of the transgenic cells. The total RNA which from transgenic mouse epidermal cells and cashmere goat fetal fibroblast were extracted, and identified by reverse transcription PCR, proved that the expression results of exogenous gene8S were positive in both two kind of transgenic cells.3.8S transgenic mouse embryonic stem cells have been obtained and preparation of transgenic miceTransgenic mouse embryonic stem cells positive clones which exogenous gene8S has been stably integrated into the genome have been obtained by transfection, lay the foundation for the preparation of chimeric mice. Simultaneously67transgenic mice were generted by pronuclear microinjection technology that injected pCDsR-K8SP into fertilized eggs of mice, in which6mice were identified as positive by PCR, the positive rate is of8.96%. In this study, preparation and identification of transgenic mice for8S laid the foundation for the further investigate properties of synthetic spider silk proteins, so for the use of bio-engineering methods to develop spider silk as a new material.