精子载体法制备转基因动物的研究进展

精子载体法是一种低成本、简单快速制备转基因动物的方法.外源基因主要通过两种方式实现基因整合,一种是直接整合到精子的基因组中;另一种是通过精子携带进入卵细胞,然后整合到受精卵的基因组中.尽管现在已经有很多利用精子载体法获得了转基因动物的研究,但是基于精子在受精过程中,结合、内化、以及转运外源基因等方面能力的差异,转染效率仍有待提高.就利用精子载体法制备转基因动物过程中,精子载体法制备转基因动物的分子机制和方法方面进行系统的阐述和分析.     

以精子为载体的体外转基因猪胚胎研究

去精清精子与外源DNA共孵育,使其携带外源DNA,再与成熟卵母细胞进行体外受精,生产转基因猪胚胎,为通过胚胎移植生产转基因猪奠定基础。结果:通过PCR从不同时期胚胎中检测出携带外源DNA的阳性胚,说明精子与外源DNA共孵育能使精子携带外源DNA,并通过体外受精技术使外源DNA进入早期胚胎。     

采用高分子介导精子作载体制备转基因泥鳅

为探讨树形高分子介导精子载体技术产生转基因动物,将泥鳅精子与具有标记基因LacZ的pCH110重组质粒和树形高分子在保存液内孵育,经DNA原位杂交检测发现树形高分子介导下精子携事外源DNA的效率得到较大的提高,将捕获了外源DNA的精子,再与泥鳅卵进行体外人工受精。由此发育的鱼苗经PCR和LacZ组织化学检测,获得了高比例的转基因泥鳅,外源基因LacZ在泥鳅幼苗头部得到了明显表达。     

精子载体法转基因动物的分子机制与制备策略

精子载体法转基因由于具有简便易行、对胚胎发育影响小的特点而成为最具诱惑力的转基因方法之一,因此研究者们在不同的水平上对该方法进行了研究。从分子水平上简要分析了探讨精子结合外源DNA及其内化转运的机制、外源DNA在精细胞内的存在和降解;基于各种理论研究的进展总结了精子载体法制备转基因动物的策略。     

转基因鱼研究进展

自1985年转基因鱼(Transigenic fish)研究在我国首次报道以来,转基因鱼研究有了很大进展.本文对国内外转基因鱼研究作一全面概述,供有关研究者参考.转基因鱼研究的简况从公开发表的文献看,已对13种(品种)鱼进行过转基因试验.研究目的涉及四方     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

美洲鲽抗冻蛋白基因的亚克隆及构建转基因鱼的表达载体

用限制性内切酶Ｐｓｔ Ｉ部分消解含有美洲鲽抗冻蛋白基因的ｐＣＴ５质粒,分离得到３２４ｂｐ的片段,将此片段亚克隆到ｐＵＣ１９质粒中,筛选到ｐＵＣ－ＡＦ重组子。从ｐＵＣ－ＡＦ重组子中,用ＢａｍＨＩ和ＨｉｒｄⅢ切下３２４ｂｐ的抗冻蛋白基因,再重组到含有ＳＶ４０病毒启动子的ｐＫＳＶ－１０的载体中,构建成转基因鱼的表达载体,此表达载何可用于鱼的基因转移的研究。     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

人生长激素基因导入金鱼受精卵内的整合、表达和促生长效应研究

通过显微注射法把小鼠ＭＴ启动子与人生长激素的融合基因（ＭＴ－ｈＧＨ）导入红龙睛金鱼受精卵内,共注射１５０６个卵子,获得８００多尾成鱼。经斑点和Ｓｏｕｔｈｅｒｎｂｌｏｔ分子杂交表明,导入的ＭＴ－ｈＧＨ基因与部分受体鱼的染色体ＤＮＡ发生了整合,整合率为２２％,转基因鱼血清中的生长激素含量平均高于对照组,平均生长速度高于对照组。导入的ＭＴ－ｈＧＨ基因可以遗传给后代。     

Sperm motility in turbot,Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics

Synopsis Turbot sperm motility is observed using dark field microscopy and stroboscopic illumination combined with video recording. Sperm motility is triggered by dilution of spermatozoa in sea water or in non ionic media (glucose or saccharose), presenting osmotic pressure ranging from 300 to 2100 mOsmol. The percentage of motile spermatozoa reaches 100% under conditions of osmotic pressure of 300 to 1100 mOsmol and pH close to 8.0. In full sea water, glucose or saccharose solutions an agglutination of spermatozoa is observed; this is prevented by addition of bovine serum albumin (5 mg ml^–1). Immediately after transfer in activation solutions, 100% spermatozoa are motile in most samples freshly stripped. This percentage drops suddenly between 15 and 30% after 70 to 100 sec. The beat frequency remains at a constant value of 50 Hz during 40 s post activation and then drops suddenly between 15 and 30 Hz. The spermatozoa velocity is about 200 micrometers s^–1 during 30 to 40 s and then declines to a stable value of 100 micrometers s^–1 at 50 s post activation. After 1.20 mn, more and more spermatozoa become motionless. The minimum calculated and averaged distance covered during 1.20 min, is about 12 mm. The high performances of turbot spermatozoa motility are interpreted as a compensatory mechanism for the low sperm production.     

Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-dried Sperm in Eggs of Nile Tilapia

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.     

精子DNA损伤检测技术的研究进展

精子DNA完整性与男性生育力之间的关系是近些年来生殖医学研究领域的热点之一,精子DNA损伤已成为反映男性生育力的一个新指标。精子DNA的损伤原因有很多,有时可能是多种因素共同作用的结果。生殖系统疾病、环境污染、吸烟、微量元素及各种理化因素等原因都可能导致精子DNA完整性受损。常见的精子DNA完整性检测技术有原位末端标记法、精子染色体扩散实验、精子染色质结构分析试验、单细胞凝胶电泳、荧光原位杂交技术和8-羟基脱氧鸟苷测定法等。随着检验技术的不断发展,关于精子DNA损伤的检测技术也在不断更新改进。本文主要就近十年来精子DNA损伤机制、检测技术的相关研究进展作一综述,提示现有的精子DNA完整性检测技术尚不能满足临床和科研需要,急需找到一种理想的检测方法为男性不育的诊断和治疗提供重要依据。     

腺病毒载体在疫苗研究中的应用

以病毒为载体的活疫苗为疾病预防和治疗研究提供了新手段。目前用于疫苗研究的病毒载体主要包括痘苗病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体及逆转录病毒载体等。其中,重组腺病毒载体因其基因组大小适中,易于基因重组操作,繁殖滴度高,易于大量制备和保存,宿主范围广,转导效率高,安全性好,能刺激机体产生强烈的体液和细胞免疫反应等特点,而被广泛应用于重要感染性疾病及恶性肿瘤的疫苗研究。腺病毒载体在人免疫缺陷病毒(HIV)疫苗研究和临床试验中的成败更是备受关注。然而,与其他载体疫苗一样,机体对载体的免疫反应仍是阻碍腺病毒载体疫苗在临床中广泛应用的主要问题。那么,腺病毒载体解决这类问题的优势何在?我们简要综述腺病毒载体的特点及其在疫苗研究中的应用和存在的问题,为进一步优化和利用腺病毒载体在疫苗方面的研究提供参考。     

TAT蛋白介导外源物质进入细胞的作用机制探讨

来源于人类免疫缺陷病毒HIV-1的反式激活因子(trans-activator,TAT)蛋白能够有效的介导多肽、蛋白质、基因以及一些其它的物质进入细胞。它以低亲和力与细胞上的受体相结合,通过破坏细胞质膜,以非内吞途径转导外源物质进入细胞内部。     

哺乳动物细胞高效表达载体的优化

目的:优化哺乳动物细胞表达系统,提高目的基因的表达效率。方法:以组织型纤溶酶原激活剂(tPA)为报告基因,利用本实验室建立的CHOfrt/dhfr-细胞定点整合表达系统,对多种表达调控元件(包括hCMV和hEF-1α启动子、hCMV增强子、hEF-1α1st内含子及翻译增强子H213和V163等)及其多种组合的表达效率进行了系统的比较和评价。结果:hCMV启动子与H213组合以及hEF-1α启动子与V163组合的表达效率分别是仅含hCMV启动子的156.6%和139.5%。结论:该研究为构建高效的哺乳动物细胞表达载体奠定了基础。     

Study on Organelle DNA Within the Generative Cell and Sperm Cells in Pharbitis

The organelle DNA in generative cell and its behavior during spermatogenesis in Pharbitis limbata and P. purpurea were observed by epifluorescence microscopy stained with 4',-6-diamidino-2-phenylindole (DAPI). In these two species, the generative cell is long and thin in which a great amount of cytoplasmic DNA is present. Most pairs of sperm cells are isomorphic, in which one end is obtuse and the other is elongate, but in a few pairs dimorphi sperms are present. The nucleus is located at one end of the cell. A lot of cytoplasmic DNA are distributed randomly throughout the cytoplasm. The size of organelle nucleoids and their fluorescence intensity are different in a sperm cell. The features of generative cell and sperm cell, and behavior of cytoplasmic DNA are similar in P. limbata and P. purpurea. The obvious differences between them are that the size and fluorescence intensity of organelle nucleoids in P. purpurea are respectively smaller and weaker than in P. limbata. The results showed that morning glory has potential of biparental or paternal cytoplasmic in heritance. Isomorphism and dimorphism of sperms, and the relationship between the ratio of nucleus and cytoplasm in sperm cell and the plastid biparental inheritance are discussed.