Construction Of A Lentiviral Vector To Target Expression Of S100A4Gene Of Antlerogenic Periosteal Cells Of Sika Deer(Cervus Nippon)

For the past few years, deer farming has been expanding quickly all around the world like China,New Zeland, Canada, Russia and so on. In Europe and USA, they mainly produce venison. In SoutheastAsia, such as China, Korea, Japan, they mainly harvest deer antlers as one of important Chinesemedicinal materials for treatment or nourishment. Deer antlers, which are the unique mammalian organs,can cast and fully regenerate year after year. The unprecedented growth rate of antlers can reach1~2cmper day. The growth rate of tumour cells is also very fast. However, antlers will not become cancerous.Therefore, we can take the deer antler as a good biomedical research model.S100A4protein is the short peptide, which consists of101amino acids. This gene does not expressin the normal tissue, but it expresses highly in cancer tissue. At present, some report has confirmed thatantler regeneration is a stem cells-based process, and that its stem cells are antlerogenic periosteal cellsand pedicle periosteal cells. Gel dimensional electrophoresis and western blot experiment had revealedthat S100A4gene expresses in the antlerogenic periosteal cells while not in pedicle periosteal cells.Pedicle periosteum is derived by antlerogenic periosteum directly. After formation of a pedicle,antlerogenic periosteum would not be distinguished. According to this particular phenomenon, wespeculate that S100A4is likely to play a leading role in antler generation and regeneration. Therefore, toresearch the function of S100A4deeply will be beneficial to the further study in the underlyingmechanism and regulatory system of antler regeneration, and be of great significance to regenerationmedicine and cancer control or even treatment.Based on the small interfering RNA design criteria reported in the related papers, we screenedsome high score sequences targeting S100A4gene of sika deer. To confirm suitability among them byBLAST research in the web named National Center of Biotechnology Information (NCBI). Then, wedesigned the corresponding short hairpin RNA sequences, and synthesized them. After annealing thesense and antisense strands of short hairpin RNA, we ligated them into the lentiviral plasmidsnamed pLVTHM which had been digested by Cla Ι and Mlu Ι firstly. Then, positive clones wereidentified based on the results of both PCR and sequencing. The positive recombination clones,pMD2.G and pCMV-dr8.91were all extracted and purified by endotoxin free maxi kit. Then, theywere co-transfected them into293t cell line by liposomes. We observed the effects24hours afterco-transfection. If observing enough green fluorescent, we could collect and concentrate virussupernatants. Then, a part of them were infected antlerogenic periosteal cells. After48hours, weobserved the effects under inverted fluorescent microscope.This study successfully identified two high score sequences targeting S100A4gene of skia deer.After annealing, the synthesized double chains short hairpin RNAs were inserted into the carrierplasmids. Firstly, we preliminarily tested they were connected successfully with digested pLVTHMby PCR. Then, we affirmed by sequencing. After293T cells were co-transfected by three endotoxinplasmids for24hours, we observed numerous of green fluorescent protein under invertedfluorescent microscope. After antlerogenic periosteal cells were infected for96hours, we also observed numerous of green fluorescent protein. The results showed that we had successfullyconstructed recombinant lentiviral system targeting sika deer S100A4gene. Moreover, we gothigh-efficient concentrated virus supernatants. These works laid the foundation for silencingS100A4gene in vivo.