| (1)Spermatogenic cell (spermatogonial stem cell) is the only cell type in postnatal mammals which has the capability to self-renew and to contribute genetic information to the next generation. The manipulation of spermatogenic cells and modification of their genomes have great significance in treatment for male sterility, gene therapy by germ cells as well as building transgenic animal model. With this assay we analyzed the possibility of Effectene TM reagent mediated gene transferring into spermatogenic cells in vitro. The efficiency of it was compared with other methods. The effect of transplantation with different time schedules on the transfection efficiency and gene expression was also investigated. Based on the development of transfection of spermatogenic cells by Effectene TM reagent in vivo, the transgenic mice with human coagulation factor IX was established.1. We compared the possibility and efficiency of transfection of mice spermatogenic cells by different methods in vitro including Effectene TM reagent, electroporation, liposome, calcium phosphate etc. The results showed that Effectene TM reagent could help to introduce exogeous gene to spermatogenic cells in vitro. Although the efficiencies observed from all methods are lower than normal haploid cells, the Effectene TM reagent showed more efficiency and lower damage to cells.The spermatogenic cells transfected by Effectene TM reagent were then transplanted to seminiferous tubule of recipient mice treated by Busulfan.2. Spermatogenesis rebuilding, normal proliferation and differentiation and gene expression were found in seminiferous tubules. Exogenous gene was detected in the epididymis genomic DNA of the recipient mice by PCR.3. The different time schedule of transplantation has great impact on the number of the positive cell clones, optimal time for transplantation should be within 12 hours after transfection. Combined with optimized transfection technique mediated by Effectene TM reagent, it offers a promising tool for production of transgenic animal model. 4. The efficiency of transfection of spermatogenic cells in vivo by seminiferous tubule injection of EffecteneTM reagent: DNA was higher than the result from testis interstitial injection method. 5. Building of human coagulation factor IX transgenic mice by injecting Effectene TM reagent: DNA (hFIX) complex into the seminiferous tubules of mice. After detection by PCR and Southern, 2 mice whose genome integration with hFIX expression element were found within 41 progenies, the efficiency of integration was 4ï¼…(2/41). One of the 2 mice expressed hFIX protein at 4.6ng/ml (detected by ELISA)(2)Flt3 ligand (fms like tyrosine-3 ligand,FL)is a membrane-bound type hemopoietic growth factor which discovered after SCF and GM-CSF. It widely distributed in hemopoietic interstitial cells, kidney, liver, placenta and other tissues with different isoforms. FL could greatly promote the proliferation,differentiation, mobilization and rebuilding of hemopoietic progenitor/stem cells when synergized with other growth factors. Meanwhile FL could significantly stimulate the proliferation of lymphocytes especially DCs and NK cells. FL has wide and promising potential in clinic therapy of some infectious disease, tissue transplantation as well as cancer therapy. To produce FL by the mammary gland bioreactor of transgenic animal, we cloned the fragment from outside-membrane region and expressed hFL both in vivo and in vitro. Mammary gland tissue specific expression vectors for human FL gene were constructed and tested in human mammary gland tumor cells.1. 537bp human FL gene fragment was cloned by RT-PCR from total RNA of peripheral blood mononuclear cells (PBMC) and eukaryotic expression vector was constructed. Detection of the expression of pCDNA3K3 in BHK cells showed that the transient expression level is 5.7ng/mL. Western result demonstrated that 537bp human FL gene fragment could express human FL correctly.2. After injection the plasmid pCDNA3K3...
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