精子介导的人凝血因子Ⅸ转基因绵羊技术研究

【作者】 李峰；

【导师】 薛京伦；

【作者基本信息】 复旦大学 ， 遗传学， 2005， 博士

【摘要】 自从1989年Lavitrano通过精子介导的转基因技术（SMGT）获得了转基因小鼠以来,大量的证据表明精子细胞还存在一个长期不被人们所知的特点:即它们可以自发的吸收外源DNA。尤其最近Lavitrano（2002）等又通过这种SMGT方法产生了大量用于异种器官移植研究的hDAF转基因猪,效率明显高于其他方法。本研究在借鉴以前精子介导的转基因技术研究结果的基础上试图剖析并阐明其基本的分子机制,为最终有可能建立一套适合绵羊的精子介导的转基因技术程序,简化转基因绵羊产生的过程奠定扎实的理论基础。本研究首次探讨和初步确定了公羊精子供体的筛选程序。首先利用绵羊常规育种参数（射精量、精子密度及精子畸形率、精子活率）,尤其注重候选公羊原精的平均活力、两次稀释、冲洗后及在17℃的水浴中温育2H后的平均精子活力对21只候选公羊进行初步筛选,然后再检测了公羊精子膜结构和功能的完整性（ORT和SYBR-14/PI）,最后比较了不同个体精子对外源DNA的不同浓度的反应,最终从21只公羊中确定了2只公羊作为精子供体。本研究首次探讨了绵羊精子介导的转基因技术的分子机理。比较了精子在不同的离子条件下(SFM和TALP+Ca²⁺、TALP—Ca²⁺)中的超激化运动;进行了内化入精子核中的质粒DNA拯救实验;进一步研究了精子同外源DNA之间的相互作用,比较了不同的离子条件下不同DNA构建体的分裂模式,为进一步研究SMGT的分子机理提供有益的参考。研究结果表明精子存活时间较长的个体,ORT、SYBR/PI这两项辅助指标也较高,在选择精子供体时它们均可以作为主要的参考指标。公羊温育8H后精予活力的高低同精子吸收外源DNA的能力有关,公羊精子活力越高,精子吸收外源DNA的能力越强。有关SMGT分子机理的研究结果首次揭示在含Ca²⁺⁺的TALP液和不含Ca²⁺⁺的SFM液中核酶均被激活,EDTA没能够阻止外源DNA内化所引起的精子自身染色体降解的代谢活动;核酶活性的激活同外源DNA序列有关。环状结构更容易激活核酶活性,线性化的结构激活核酶活性依赖于外源DNA的结构,pBluescript SK（+）较pDNA3更易激发精子内核酶活性;没有发现绵羊精子内核酶活性强弱同外源DNA添加量相关;精子内核酶激活后裂解外源DNA的同时也裂解自身的染色体,激活的核酶似乎不只一种,不同结构的环状质粒激活了不同的核酶,这些核酶在精子基因组上存在不同的作用位点;选择合适的外源DNA序列对精子介导的转基因技术的成功率至关重要。最后我们有理由假设,精子也许存在某种机制来阻止来自精子DNA损伤所产生的危害向胚胎的传播。在受精时期,如果一个精子细胞遇到一个潜在的DNA损伤环境,它也许能够通过一种特异性的精子自杀行为作出反应,即消化自身DNA成圆环状大小的DNA,进而不具备受精能力。 进一步结合绵羊体外生产胚胎技术对精子介导的转基因技术的分子机理进更多还原

【Abstract】 Since 1989 Lavitrano described the binding of exogenous DNA in mouse, evidence has accumulated that clearly indicates that spermatozoa have the surprising property of the allowing the intemalization of molecules of different size and nature, including DNA. A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer （SMGT）. The efficiency of transgenesis obtained with SMGT was much greater than with any other method （Lavitrano et al., 2002）. On the basis of the results of previous SMGT, we try to explore the prescreening parameters of sperm donor in sheep, analyze the molecular mechanism of SMGT in sheep, our study will be of benefit to those whose work requires the use of transgenic large animal models in medicine and animal biotechnology. On the first time, the study explored and established the procedure of prescreening of sperm donor in ram. Standard parameters used in conventional animal breeding programs （volume, concentration, presence of abnormal sperm cells, and high progressive motility） were used to prescreen 21 Ram, mainly focusing on motility at time of collection, high progressive motility after 2 hr. We performed an osmotic resistance test to evaluate the integrity of membrane function and assessed sperm membrane integrity by SYBR-14 and propidium iodide （SYBR-14/PI）. Finally another important parameter evaluating to optimize the amount of exogenous DNA per spermatozoa, which is whether sperm is resistant to increasing concentrations of DNA, was used to select the donor of sperm. In this study, 2 of 21 rams studied were used as sperm donors. Molecular mechanism of SMGT in sheep was explored. HAM of ram sperm was studied among the SFM, TALP+Ca²⁺ 、 TALP free of Ca²⁺, the fate of the foreign DNA internalized in ejaculated spermatozoa in sheep was tracked. Our results showed that the sperm nucleus is not a safe place for exogenous DNA since its intemalization activates a metabolically active process similar to apoptosis which destroys both the DNA and the sperm cell. The endogenous nucleases in ram spermatozoa, which may cause degradation of DNA at hypersentive sites of sperm chromosome under certain condition （the different foreign DNA）, were activated in the medium either containing Ca2+( TALP+Ca²⁺) or free of Ca²⁺ （SFM）. The activation of endogenous nucleases in ram spermatozoa was associated with the sequence and conformation of the foreign DNA, EDTA failed to prevent foreign DNA-dependent chromosomal damage, the endogenous nucleases was not activated in a dose-dependent. Those results indicated that the suitable sequence of foreign DNA was important for the higher efficiency of SMGT. It is reasonable to predict that spermatozoa would have some更多还原