Study Of CD320 And RanBP9 Expression In Goat Spermatogonial Stem Cells

Sperm-mediated gene transfer(SMGT) is an effective and simple method for transgenic animal generation, yet the mechanism is still unclear. Our previous studies have shown that CD4 plays major roles in goat sperm internalizing exogenous DNA; through using yeast two-hybrid system, we screened new candidate molecules, CD320 and RanBP9, interacting with CD4; furthermore, we confirmed the interaction between CD4 with CD320 and RanBP9 by co-immunoprecipitation. However, the role of CD320 and RanBP9 in sperm transfection of exogenous DNA is rarely investigated. As animal sperm is originated from metamorphosis and highly differentiation of sperm cells, it factually unable to proliferate and differentiation, which it is therefore a bad case to study genes’ function.Spermatogonial stem cells(SSCs) originate from spermatogenesis after gender differentiation and are the basis for sperm generation and male fertility. SSCs have the capabilities to maintain a constant number of germ cells by self-renewal and to produce spermatocytes via direct differentiation, as well as are the unique adult stem cells which delivery animals’ genetic information to the next generation. SSCs have strong plasticity that can be reprogrammed into embryonic stem cell-like pluripotent stem cells or be induced to differentiate into sperm-like cells, as a result, it become the ideal seed cells for genetic and cell engineering in vitro. Recently, studies about SSCs culture in vitro of mice, humans, cows and other animals has been widely investigated, however, regarding to SSCs of goat is rarely reported.In this study, we firstly separated, purified, and identified goat SSCs, and then cultured goat SSCs in vitro. Then we transfected exogenous CD320 and RanBP9 plasmid into goat SSCs to investigate the gene expression of CD320 and RanBP9 in goat SSCs, providing thesis foundation of CD320 and RanBP9 in sperm transfection of exogenous DNA lay the foundation. The main results follows:1. Separation, purification and identification of goat SSCs. We collected 4 months old goat testicles, and isolated testicular cells by two-step enzyme digestion method, and then obtained single cell suspension via differential adhesion method. Purified spermatogonia were cultured with no feeder layer, 1% serum and GDNF, LIF, bFGF at 37℃, 5% CO2. We observed the growth state of cells, and then identified cells by alkaline phosphatase, immunocytochemistry staining and RT-PCR. The obtained results showed that cells had generated remarked colony at 14 days, and still formed cell clones after subculture with decreased the hybrid cells surrounding of clones; when clone passaged to the second passage, cell clones decreased, but shuttle type cell increased. Alkaline phosphatase staining showed that the cells of different time had different density, but not somatic cell. Immunohistochemical staining showed that cell clones expressed SSCs markers, PGP9.5 and PLZF, but not somatic cell; the results from RT-PCR showed that the mRNA abundance of SSCs specific markers, CD29, CD49 f, PLZF, OCT-4 and THY1, were significant higher than that of somatic cells, but the mRNA abundance of P21 in SSCs was significant lower than that of somatic cells. Collectively, the obtained results verified the cell clones were SSCs clone and could conduct following experiments.2. CD320 expression in goat SSCs. We designed specific primers for CD320 and detected the mRNA abundance of CD320 of SSCs in goat by real time PCR. Then, we constructed goat CD320 vector to detect the location of CD320 in 293 T cells and transfection in goat SSCs. The results from RT-PCR showed that the mRNA abundance of CD320 in SSCs were significant higher than that of somatic cells. Cell transfection showed that CD320 was located at the cytoplasm of 293 T cells and in SSCs clones. However, when SSCs were subcultured, CD320 failed to express.3. RanBP9 expression in goat SSCs. We designed specific primers for RanBP9 to detect the mRNA abundance of RanBP9 of SSCs in goat by RT-PCR. Then, we constructed goat Ran BP9 vector to detect the location of RanBP9 in 293 T cells and transfection in goat SSCs. The results from RT-PCR showed that the mRNA abundance of RanBP9 of SSCs is higher than somatic cells, but the difference was not significant. Cell transfection showed that RanBP9 was located at the whole 293 T cells and goat SSCs clones. When goat SSCs were subcultured, we found that RanBP9 located into the cytoplasm of SSCs. Cell proliferation assay showed that cell proliferation of SSCs transfected RanBP9 was higher than that of un-transfected SSCs.