| Spermatogoanial stem cells are the only adult stem cells which can pass their gene to the next generation, retaining the potention of self-renew and differentiaton. With the appearing of spermatogonial stem cell transplantation technology, the biological functions and applications researches about spermatogonial stem cells have become new focus. Compared with the traditional methods of preparing a transgenic animal, preparing transgenic animals by spermatogonial stem cells transplantation has a wider range of advantages. Such as easily obtained, Cell lines is more stable during vitro procedure and the higher transgenic efficiency.Successful preparation of transgenic animals by spermatogonial stem cell transplantation technology depends on several factors, including:1. The technology of culturing spermatogonial stem cells in vitro and genetically modification of spermatogonial stem cells.2. Recipient preparation technique which try the best to remove the endogenous spermatogonial stem cells, under the premise that the spermatogonial stem cell nich of the recipient is from destruction.3. Particular spermatogonial stem cell transplantation method which is suitable for a particular species. Rats are mainly studied in this paper, long-term culture of rats spermatogonial stem cells technology have been established by our group, based on that technique, we explore specific methods of genetically modified of spermatogonial stem cells.1. Culture and genetically modified of rat spermatogonial stem cellsBased on the technology of long-term culture of rat spermatogonial stem cells which have been established by our group, we explore the appropriate method to modify the rat spermatogonial stem cells by lentiviral. The rat spermatogoial stem cells had been identified by immunofluorescence staining, to ensure that the characteristics of the spermatogonial stem cells do not have been changed. The promoter of the lentivirus is EFla, carrying the EGFP reporter gene. We observe the morphology of spermatogonial stem cells and feeder cells every2hours, during the6th and15th hours. Determine the best infection time of this lentiviral to rat spermatogonial stem cells preliminary. Based on the research above, We adopt four infectious dose to infect the spermatogonial stem cells, respectively MOI=5、MOI=10、MOI=30、 MOI=60. After24and48hours, observe the morphology and expression of green fluorescent protein of the spermatogonial stem cells by fluorescence microscope. The best MOI of this lentiviral to rat spermatogonial stem cells was obtained by the research.2. Preparing rat spermatogonial transplantation recipient by treating the pregnant female rats with busulfanSuccessful SSC transplantation is largely dependent on the preparation of the recipient, which involves the destruction and blockade of endogenous germ cells and spermatogenesis to allow donor SSCs to translocate from the lumen to the basal compartment of the seminiferous tubules and begin donor-derived spermatogenesis. Although busulfan, a sterility-causing drug, is commonly used for the preparation of the SSC transplantation recipients, in the rat, new technology has been required to acquire more efficient recipients. In the present study, the response of pregnant female rats to treatment with7.5-12.5mg busulfan per kg-1of body weight was determined in terms of the birth rate, survival rate, body weight, testicular mass, histology, expression of certain germ cell genes as measured by real-time PCR and fertility rate of male offspring. The analysis suggested that a dosage of10mg/kg of busulfan administered to pregnant female rats at13.5days post pregnancy effectively prepares male pups to be recipients because it leads to the maximal deletion of endogenous spermatogonial stem cells with minimal effects on the recipients’health. This dosage can create sufficient niches and maintain the microenvironment for sufficiently longer time periods so that colonization of donor-derived spermatogonial stem cells can take place.3. Explore the optimal method of rat spermatogonial stem cells transplantation.1994, Brinster created the spermatogonial stem cells transplantation technology, which received extensive attention. Playing an important role in promoting the study of spermatogenesis, spermatogonial stem cell transplantation is with great value in the applications, such as,1. Preservation of the fertility of males,2. Treatment of male sterility,3. Preparation of transgenic animals. Not only the preparation of recipients, but also the transplantation method has an important influence on transplantion outcomes. Though the spermatogonial stem cells transplantation methods had been discussed many times in mouse, with the different anatomical structures to mouse, the special method for the spermatogonial stem cells of rat should be made a thorough research. In this study, we start from the preparation of the glass needle and the microinjection methord, an effective rat spermatogonial stem cells transplantation method have been established.4. The established of the plasmid vector which could detect the completion of meiosis in the male.Maturation of male germ cells in mammals involves numerous structural and functional changes that are precisely timed. These complex processes, known collectively as spermatogenesis, may be represented by the following three major events:proliferation and differentiation of spermatogonia, meiotic events at prophase of spermatocytes, and drastic morphological change during differentiation from the haploid round spermatids to sperm.。To the best of our knowledge, research on mammals spermatogenesis is undergoing slow-moving development.Genetic engineering is needed to mark important events during the spermatogenesis. Gsg2was especially useful for detecting the completion of meiosis.We intend to constructe CMV-eGFP-Gsg2-DsRed vector which can start the expression of EGFP in all transgenic cells and start the expression of DsRed in the germ cells that have completed the meiosis proceed. The recombinanted vector is based on the CMV-eGFP-DsRed vector which is conserved in our labrary, and the Gsg2promotor is inserted into the MCS. Via the transfection test to the rat testis co-culture cells, proof the construced of CMV-eGFP-Gsg2-DsRed vector which contain the Gsg2promotor is successful. |
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