精子介导的转基因效率关键影响因素

Research on Key Factors Restricting Efficiency of Sperm-Mediated Gene Transfer

【目的】精子介导的转基因技术简单易行,其作为制备转基因动物的一种方法已经被很多科学家所认同。但是不同实验室的研究结果显示其稳定性和重复性较低,转基因效率差异极大。现将主要探讨导致这种现象的原因。【方法】小鼠精子获能后分别与0、15、30和300nmol·L-1的Cy-3标记DNA(Cy-3-DNA)在37℃共孵育30 min。其后,血细胞计数板检测小鼠精子的活率;共孵育精子进行直接涂片,DPBS洗涤后涂片,DnaseI消化后涂片,精子涂片置于荧光显微镜下,记录视野中的精子总数及显示Cy-3信号的精子数,用于统计精子结合及内化DNA的效率。精子与300 nmol·L-1的Cy-3-DNA共孵育后,以50μmol·L-1的P4诱导精子顶体反应,统计顶体反应前后显示Cy-3信号的精子比例。精子分别与15和300 nmol·L-1 Cy-3-DNA共孵育后,精子进行体外受精,在荧光镜下观察合子期受精卵中Cy-3-DNA的存在位置。按照优化后的条件,精子分别与0、15和300 nmol·L-1 pEGFP-C1质粒共孵育后进行体外受精,制备转基因胚胎。比较试验组和对照组的受精率和发育效率,采用PCR和RT-PCR方法检测囊胚期外源基因整合和表达情况。【结果】获能后的精子分别与0、3、15、30和300 nmol·L-1的Cy-3-DNA共孵育后,精子的活率分别为82.21%、73.63%、77.38%、76.33%和77.80%;洗涤前精子结合DNA的效率分别为76%、94%、99%和100%,15、30和300 nmol·L-1组精子结合率均显著高于3 nmol·L-1组(P<0.05)。洗涤后精子结合DNA的效率,分别为45%、66%、84%和87%,消化后精子内化DNA的效率分别为44%、56%、71%和76%,并且洗涤后精子结合及消化后精子内化DNA的效率都为300 nmol·L-1组和30 nmol·L-1组均显著高于其他两组,而15 nmol·L-1组显著高于3 nmol·L-1组(P<0.05);Image J对精子结合Cy-3-DNA的强度进行分析的结果表明,外源DNA的量随着外源DNA浓度的增加而显著增加(P<0.01)。顶体反应后,精子顶体部DNA会有丢失,但精子头部后区的外源DNA依然会保留,因此,顶体反应前后,精子结合外源DNA的精子比率没有降低。15和300 nmol·L-1 DNA共孵育的精子体外受精后,荧光显微镜下检测显示,合子期胚胎有外源DNA的荧光信号分布,将荧光信号在雄原核附近密集的胚胎记为阳性胚胎,300 nmol·L-1 DNA共孵育的精子获得合子期胚胎的阳性率为27.89%,显著高于15nmol·L-1组的9.00%(P<0.05)。精子与pEGFP-C1质粒共孵育后,体外受精,结果表明精子与DNA共孵育后,卵母细胞受精率及胚胎卵发育率与对照组相比差异不显著。单囊胚PCR检测15和300 nmol·L-1的pEGFP-C1质粒与精子共孵育转基因胚胎阳性率分别为8.72%和21.50%;采用RT-PCR方法在300 nmol·L-1组的囊胚中检测到了EGFP基因RNA的表达,在荧光显微镜下没有观察到EGFP绿色荧光蛋白的表达。【结论】精子具备结合和内化外源DNA的能力,并能将外源DNA带入卵母细胞,顶体反应会使精子丢失顶体部DNA,但精子头部后区外源DNA依然保留。受精过程外源DNA在基因组中的整合及外源基因表达激活是精子介导转基因效率的关键影响因素。

[Objective] Sperm mediated gene transfer technique is simple, it has been recognized by many scientists as a way to produce transgenic animals, however, the results from different laboratories showed poor stability and reproducibility, transgenic efficiency is also significantly different. The aim of this study is to clarify the main factors causing this phenomenon. [Method] Capacitated sperms were incubated with 0, 15, 30 and 300 nmol.Ll Cy-3 labeled DNA （Cy-3-DNA） at 37℃ for 30 min. After incubation, sperms viabilities were detected by CBC board. Sperms incubated by DNA were used to direct smeared, smeared after washing by DPBS and smeared after digested by Dnase I, sperm smears were placed under fluorescent microscope. The total number of sperms and the number of sperms which show Cy-3 signal in the vision were recorded, the records were used for the efficiency statistics of sperms binding and uptaking exogenous DNA. 501~mol-L1 P4 were used for inducing sperms acrosome reaction which were incubated with 300 nmol.L-1 Cy-3-DNA. The proportion of sperms showing Cy-3 signals were checked before and after the acrosome reaction. Sperms incubated with 15 and 300 nmol.L-1 Cy-3-DNA were used for IVF, the presence of Cy-3-DNA in the zygotes was detected under fluorescence microscopy. According to the optimized conditions for these experiments, sperms incubated with 0, 15 and 300 nmol.L1 pEGFP-C1 were used for getting transgenic embryos, and oocyte fertilization rate and embryonic development rate in experimental group and control group were compared, PCR and RT-PCR were used to detect the presence and expression of pEGFP in the blastocyst. [Result] Sperms after capacitation were incubated with 0, 30, 15, 30 and 300 nmol.L1 Cy-3-DNA, the sperm viability was 82.21%, 73.63%, 77.38%, 76.33% and 77.80%, respectively. The rate of positive sperms was 76%, 94%, 99% and 100% before washing, and that in 15, 30 and 300 nmol＇Ll treatment groups had more efficiency than 3 nmol＇L1 （P〈0.05） group. The rate of positive sperms was 45%, 66%, 84%, 87% after washing and 44%, 56%, 71%, 76% after digestion, and that in 300 and 30 nmol＇L-l treatment groups had more efficiency than the other two groups, and 15 nmol.L1 treatment group is had more effiency than 3 nmol.L-I （P〈0.05） group. The result showed that the amount of uptaking DNA increased significantly as the increasing of DNA concentration by ImageJ analysis（P〈0.01）. After acrosome reaction, DNA attached on the acrosome would be lost, but DNA exist in the rear of the sperm head still retained, so the ratio of positive sperms did not decrease. Sperms incubated with 15 and 300 nmol.Lq DNA were used for IVF. There were exogenous DNA fluorescence signals distributed in zygotic embryos under fluorescence microscopy, embryos which fluorescence signals densely distributed in male pronuclear were recorded and considered as positive embryos. The positive rate of zygotic embryos derived from sperm incubated with 300 nmol-L1 DNA was 27.89%, which was significantly higher than that incubated with 15 nmol.Ll （P〈0.05）. Sperms were incubated with pEGFP-C1 plasmid were used for IVF. The result showed that incubation did not affect the oocyte fertilization rate, and the embryonic development rate compared with the control group. Single blastocyst PCR detection showed that the transgenic blastocyst rate derived from sperms incubated with 15 and 300 nmol.L1 pEGFP-C1 was 8.72% and 21.50%, respectively. RT-PCR detection of EGFP expression in the blastocysts derived from 300 nmol.L~ DNA showed positive results but no protein expression was detected by fluorescence microscopy. [ Conclusion] Sperms have the ability of binding and uptaking exogenous DNA, and sperms can carry exogenous DNA into oocytes. Acrosome reaction causes part loss of the DNA, but in the rear zone of the sperm head, DNA is still retained. The key factors restricting the efficiency of sperm-mediated gene transfer might be integration of exogenous DNA into the genome and expression activation of the exogenous genes during fertilization.