Research On Key Factors Restricting Efficiency Of Sperm-Mediated Gene Transfer

It is simple that Spermsperm-mediated gene transfer technique is simple, itwhich has been recognized by many scientists as a way to produce transgenic animals, howeverHowever, the results from different laboratories showed poor stability and reproducibility, and the transgenic efficiency is also significantly different. This study analysised the main cause of this phenomenon.First,we conducted experiments to produce transgenic embryos by bperm-mediated gene transfer technologywhich reports commonly used technical solution. Sperm were incubated with pEGFP-C1, pEGFP-PB, pEGFP-SMAR respectily under the conditions of5μg/ml and incubated30min at37℃for further determining the actual effectiveness of the technical.It was foundthat embryos obtained after infrom vitro fertilization when in which sperms were treated with three plasmids showed normal embryonic development compared to the control group. No GFP protein expression were detected in the blastocysts by fluorescence microscopy and immunofluorescence.Then,we research on the key factors restricting efficiency of sperm-mediated gene transfer by through the study of each period of sperm-mediated gene transfer. Capacitated sperms were incubated with3-300nmol/L Cy-3labeled DNA at37℃for30min followed,digested digesting by Dnase I, and then the effect of DNA incubation on sperms viability were detected and the efficiency of sperm binding and uptaking exogenous DNA were checked under fluorescence microscopy. Acrosome reaction system was optimized that,50μmol/L P4were used for inducing acrosome reaction, and the effects of acrosome reaction on the efficiency of sperm carrying exogenous DNA were checked. Incubated sperms were used for IVF, and the presence of fluorescently labeled DNA in the zygotes were detected under fluorescence microscopy. According to the optimized conditions form these experiments, sperm incubated with pEGFP-C1were used for getting transgenic embryos, and PCR and RT-PCR were used to detect the presence and expression of pEGFP in the blastocyst. The pEGFP-C1plasmid was injected directly into the cytoplasm of zygotic embryos, for comparinge theof effect of different concentrations on the expression efficiency of GFP. Results are as follows:1. Sperm after capacitation was incubated with0、3、15、30、300nmol/L Cy-3-labeled DNA,in which, sperm viability was82.21%,73.63%,77.38%,76.33%,77.80%,respectively. The results show that when even the exogenous DNA concentration increased to300nmol/L, exogenous DNA incubation did not reduce sperm viability.2. Incubated sperms with3-300nmol/L Cy-3-DNA, and compared the incubation efficiency were compared. The results showed, that the rate of positive sperms was77%-100%before washing, and15nmol/L、30nmol/L、300nmol/L treatment group is had all more efficiency than3nmol/L. Rate of positive sperms was45%-87%after washing and44%-76%after digestion,300nmol/L and30nmol/L treatment group is had more efficiency than the other two groups, and15nmol/L treatment group is had more effiency than3nmol/L (P＜0.05). Simultaneously,the amount of uptaking DNA increased significantly as the increasing of DNA concentration by ImageJ analysis (P＜0.01).3. We used progesterone(P4) to induce sperm acrosome reaction, and detected the efficiency of acrosome reaction. Acrosome reaction conditions were optimized, and the optimum treatment was50μmol/L P4treated40min. After acrosome reaction, DNA attached on the acrosome would be lost, but the ratio of positive sperm did not decrease.4. Sperm was incubated with15nmol/L and300nmol/L DNA used for IVF.There were exogenous DNA positive signal distributed in zygotic embryos. Embryos which positive signals densely distributed in male pronuclear recorded were considered as positive embryos. The positive rate of zygotic embryos derived from sperm incubated with300nmol/L DNA was27.89%,which wassignificantly higher than incubated with15nmol/L.5. Single blastocyst PCR detection showed that the transgenic blastocyst rate derived from sperm incubated with15nmol/L and300nmol/L pEGFP-C1was8.72%and21.50%respectively. RT-PCR detection on EGFP expression in the blastocysts derived from300nmol/L DNA showed positive results but no protein expression were detected by fluorescence microscopy.6. The expression of GFP fluorescence was detected when plasmid concentration was20μ g/ml. The expression was increased as the increasing of plasmid concentration, but the efficiency of embryo development reduced.As the results above,we proved that sperms have had the ability binding and uptaking exogenous DNA, and spems can carry exogenous DNA into oocytes. Acrosome reaction causes part the loss of the part of DNA, but the key factors restricting the efficiency of sperm-mediated gene transfer is fertilization,the integration of into genome and expression activation of the exogenous genes.