Study On Establishing Transgenic Animals By Sperm-mediated Gene Transfer With Dendrimers For Xenotransplantation

Today, transgenic animals have been produced for the most part by using microinjection of exogenous DNA into the male pronuclei of zygote or other methods. Compare to others transgenic techniques; sperm mediated-gene transfer (SMGT) has two additional advantages:low cost and easy of use. However, the availability and reliability of SMGT hasn’t been approbated. In our study, we investigated the process of exogenous DNA binding and internalization to sperm and the molecular events of exogenous DNA after internalization. Our aims were to establish the platform of SMGT and use this technology system to produce GO double-gene transgenic mouse model for hHT and hDAF.Section IEstablishment of sperm mediated-gene transfer technology based on DendrimersObjective:To exogenous DNA localization and the frequency of spermatozoa carrying exogenous DNA after sperm/Dendrimers/DNA co-culture are key to a successful sperm mediated-gene transfer (SMGT). Methods:In this study, the characteristics and influencing factors of exogenous DNA uptake by spermatozoa were tested using digoxigenin (DIG) labeled DNA as trace. Results showed that mouse spermatozoa incubated with Dendrimers could spontaneously take up exogenous DNA. The exogenous DNA was initially bound to the outer sperm membrane at postacrosomal region; subsequently party of the bound DNA was internalized into nucleus. There were considerable differences in the capability of spermatozoa from different donors to bind and internalize exogenous DNA. In35samples, binding rates (before DNase I digestion) and internalization rates (the positive rate after DNase I digestion) varied between4.6%-62.4%and2.1%53.8%, respectively. For the spermatozoa from the same mouse, the binding and internalization capacities were mostly inhibited by the seminal fluid. Compared to ejaculate sperm, the binding rate and internalization rate were increased three and five times in washed sperm cells, respectively. At the same time, capacitated spermatozoa also had lower exogenous DNA uptake(P<0.01). Dead spermatozoa did not complete the internalization process. The highest positive rate (before DNase I digestion) was found in membrane-broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors. Results:These results suggest that selection of appropriate sperm donors and optimization of sperm processing procedures are the key steps for successful SMGT.section ⅡImprove the efficency of SMGT with EGTA, ATA and PMSFObjective:To explore the effect of EGTA, the specific inhibitor ATA and the protease inhibitor PMSF on sperm motility and survival time, the incidence of sperm acrosome reaction in vitro, the sperm binding ability of exogenous DNA and degradation of internalized DNA. Methods:The specific inhibitor ATA could improve sperm motility and survival time in vitro, enhance sperm binding ability of exogenous DNA, but the exogenous DNA in the cells almost completely degraded in the sperm which ATA was added. EGTA and the protease inhibitors PMSF had a good protective effect on the exogenous DNA internalizde into the sperm cells, but prevented sperm acrosome reaction. In addition, PMSF increased the ability of sperm binding exogenous DNA and enhanced the motility of sperm. EGTA had a exact opposite role.After EGTA solution and ATA were added together, the motility of sperm, acrosome reaction rates and binding ability of exogenous DNA were improved compared with which EGTA was alone added. The degradation of DNA was significantly inhibited. Results:The results suggest that in the process of sperm-mediated gene transfer, adding EGTA and ATA into the culture medium not only favorated the binding and the integrity of exogenous DNA internalized, but also ensured the fertilization of sperm.Section ⅢConstruction of hHT/GFP fusion gene expression vector and its expression in mouse fibroblastObjective:To construct the hHT/GFP fusion gene expression vector. Methods:The fusion expression vector of human alphal,2-fucosyltransferase (hHT) and (green fluorescent protein, GFP), was constructed and was expressed in mouse fibroblast. The results of PCR proved hTH was cloned and the results of restrictive enzyme digestion analysis proved the expression vector pEGFP-C1-hHT was constructed. The positive signal of GFP was observed in mouse fibroblast into which pEGFP-C1-hHT was transferred. The fibroblast with positive signal of GFP was analyzed by PCR and western blot. The integration of hHT was proven by PCR. The expression of hHT was proven by western blot. Results:These results showed that the fusion expression vector pEGFP-C1-hHT was constructed, and this vector could be used in future research work.SectionⅣ Construction of hDAF/GFP fusion gene expression vector and its expression in mouse fibroblastObjective:To construct the hDAF/GFP fusion gene expression vector. Methods:The fusion expression vector of human decay accelerating factor (hDAF) and red fluorescent protein (RFP) was constructed and was expressed in mouse fibroblast. The results of PCR proved hDAF was cloned and the results of restrictive enzyme digestion analysis proved the expression vector pDsRed2-C1-hDAF was constructed. The positive signal of RFP was observed in mouse fibroblast into which pDsRed2-C1-hDAF was transferred. The fibroblast with positive signal of RFP was analyzed by PCR and western blot. The integration of hDAF was proven by PCR. The expression of hDAF was proven by western blot. Results:These results showed that the fusion expression vector pDsRed2-C1-hDAF was constructed, and this vector could be used in future research work.Section ⅤEstablishment of Double-gene Transgenic Mouse Model for hHT and hDAF through SMGTObjective:To explore the molecular mechanism of hyperacute rejection (HAR) and delayed xenograft rejection (DXR). Methods:The double-gene transgenic mouse model expressing human alpha1,2-fucosyltransferase (hHT) and human-decay accelerating factor (hDAF) was established using sperm-mediated gene transfer optimized by dendrimers (SMGT). The positive rate of PCR for GFP+RFP in2-cell embryos was4.51%after the SMGT was optimized, which was significantly higher than that of the control group (P<0.01). Embryos expressing both GFP and RFP were got were transplanted to the pseudopregnant female mice.36offspring were got. The founder transgenic mice (GO mice) were identified with PCR and southern blot. The results of PCR showed that genomes from17mice were integrated by both hHT and hDAF. The results of southern blot for5mice showed a positive signal of hHT+hDAF. Results:These results indicated that GO double-gene transgenic mouse model for hHT and hDAF was established.