Gene Cloning, Protein Expression And Functional Analysis Of S100A4 Of Antler Stem Cells In Sika Deer (Cervus Nippon)

Deer antlers are the only mammalian appendages capable of full renewal, and therefore offer a unique model for biomedical research. Both development and regeneration of antler are come from antlerogenic periosteum. Regenerated antlers come from pedicle periosteum cells directly. We can study antler development and underlying mechanism using these two types of cells. Protein S100A4 is a cancer relative factors involved in apoptosis, metastasis, angiogenesis and so on, Analysis of the differential expression of S100A4 protein between these two kinds of periosteum cells is the enter point for my master project.In order to study the function of S100A4 in antler development and regeneration, I cultured antlerogenic periosteal cells which were subsequently used to extract total RNA, obtained the full length of the S100A4 gene through reverse transcription by Oligo-dT primer, and designed amplification primers based on bovine S100A4 gene sequence. The full length gene was then inserted into the vector plasmids pLEGFP-C1. Recombinant plasmids pLEGFP-S100A4 and the pVSV-G (envelope vector) were co-transfected into packaging cells GP2-293. The viral supernatant produced from the transfected GP2-293 was collected and used to infect the cultured pedicle periosteum cells. Active expression of S100A4 gene was integrated with pedicle periosteal cells. Selection of infected cells with G418 was carried out to gain stable express S100A4 fusion protein cell line. Expression of S100A4 gene in transcriptional level was detected by RT-PCR. Analysis the role of S100A4 protein in antler development, in particular for angiogenesis, was carried out using chick embryo chorioallantoic membrane assay.In this study, I cloned sika deer S100A4 gene for the first time, and constructed recombinant vector pLEGFP-S100, and gained a pedicle periosteal cell line that stablely express S100A4 fusion protein through G418 selection. I investigated effects of S100A4 protein on angiopoiesis. Therefore, my work has made a step forward in revealling antler development mechanism and growth regulation.