哺乳动物精子载体转基因技术新研究

精子载体法是以精子作为外源基因的载体，在受精过程中将外源基因导入动物胚胎，从而使外源基因进入子代的基因组中。相对于显微注射法、反转录病毒感染法、胚胎干细胞法等方法而言，它的主要优点是利用精子的自然属性克服了人为机械操作给胚胎造成的损伤，提高了转基因效率，而且操作简便、成本低廉。因而成为转基因动物研究的热点技术。现就精子结合外源基因的机制进行简要介绍，并对发展起来的精子细胞输精管内注射法、曲细精管徽注射法和睾丸直接注射法等精子载体转基因新途径进行综述。     

睾丸注射转基因技术研究进展

睾丸注射法将外源DNA直接注入睾丸内转染各级生精细胞,通过生精细胞的增殖分化而获得大量转基因精子,通过人工授精或自然交配生产转基因动物,是一种操作简便、成本低和适宜于大群生产的精子介导的转基因方法。本文对睾丸注射法获得转基因精子的原理以及曲精细管注射法、睾丸间质注射法和睾丸网注射法3种睾丸注射方法进行介绍,对睾丸注射制备转基因动物所应用的阳离子脂质体介导法、高分子载体介导法、二甲基亚砜(DMSO)介导法和电穿孔法、基因枪法5种转染方法进行综述,并指出了睾丸注射法存在的问题,对睾丸注射法的研究前景进行了展望。     

转基因小鼠的制备

近二十年来,分子生物学研究获得了长足的发展,而其核心部分基因工程技术的发展尤为突出,特别是转基因动物技术的出现,它不仅为人们研究外源基因在整体动物中的表达调控规律提供了最为有效的手段,而且还可通过改变动物的基因型,使其表现型更符合人类的需要,以培育优良品种及生产人类所需的有生物活性的蛋白质.因此,动物的基因转移这一生物技术已经广泛地应用于生命科学研究的众多领域.所谓转基因动物是指用实验手段将特定外源基因导入动物早期胚胎细胞,由此整合到染色体上,并通过生殖细胞系传递给后代的一类新动物.常见的转基因方法有DNA显微注射法、胚胎干细胞介导法、逆转录病毒转染法、精子载体法、电转移法等,其中以DNA显微注射法最为成熟,使用最广泛、最可靠,该法常用的哺乳动物是实验小鼠.本文所要介绍的就是显微注射法制备转基因鼠所涉及的关键技术和原理(一般程序如图1所示).     

转基因动物制备技术

所谓转基因动物是指基因组整合有外源基因,并且能将外源基因稳定遗传给后代的一类动物。本文简要介绍了转基因动物制备技术的受精卵原核显微注射法、干细胞嵌合体法、体细胞克隆法和病毒载体法四种主要技术的原理、方法及优缺点。     

精子载体转基因技术研究进展

精子因在受精过程中的独特作用,而被公认为是转移外源DNA的理想载体,且操作简便,成本低廉,危险隐患相对较低,近十几年来的研究结果表明精子载体介导法可以得到转基因阳性动物.本文对精子载体转基因方法、精子载体转基因的机理、影响精子与外源DNA结合的因素以及外源DNA在宿主动物中的存在形式等方面进行了论述,并对精子作为载体的研究方向和前景给予了预测.     

转基因动物研究进展

转基因动物技术是将已知的外源基因导入动物细胞并整合到基因组中,从而使其得以表达的技术。此技术将分子、细胞和个体水平统一起来,标志着基因工程已由离体操作发展到离体与载体相结合的新阶段。目前,转基因动物的制作方法主要有反转录病毒感染法、显微注射法、胚胎干细胞法和精子载体法等,每种方法都有其优缺点。转基因动物技术主要应用在人类疾病模型、生物反应器、异体器官移植和改良动物品种与性状等方面。同时转基因动物技术也存在一些技术难题和安全性问题,但发展前景被普遍看好。     

转基因动物制备方法

动物转基因技术是通过将外源性目的基因整合到动物染色体上,使其在体内整合和表达,获得表达外源基因动物的技术。该技术广泛应用于转基因动物育种、基因功能研究、药用蛋白生产和器官移植等方面。其主要方法包括逆转录病毒感染法、显微注射法、体细胞核移植法、胚胎干细胞介导法、精子介导法等。本文旨在对动物转基因的各种技术和方法进行综述,甄别各自的优缺点,为科研工作者提供一定的参考。     

猪基因导入研究中两种转基因方法的比较

供试受体母猪共87头,其中50头受体母猪用显微注射法获得转基因卵。37头受体母猪采用精子载体法通过人工手术输精使其受孕。其受胎率前者为63.24％,后者为30.87％;平均产仔数分别为5.38和6.18头;基因整合阳性率分别为17.9％和12.7％;转基因效率分别为2.7％和2.8％。两种方法,平均产仔数.基因整合阳性率及转基因效率之间差异不显著(P>0.05),而受胎率之间差异极显著(P<0.01)。用精子载体法导入外源基因,不需对胚胎进行体外显微操作,大大简化了生产转基因动物的程序.但此方法尚需逐步完善。     

睾丸注射Mx-NA双基因制备抗病转基因鸡的研究

本研究利用精原干细胞介导法体内转染重组载体pVITRO2-Mx-NA以生产抗病转基因鸡。采用睾丸注射法将脂质体包裹的线性化质粒通过随机打点注射入公鸡睾丸,采集25、50、60、70d后试验公鸡的精液,提取基因组DNA,采用常规PCR检测外源Mx-NA联合基因的整合;转染后60d采集阳性公鸡精液人工授精母鸡,采用PCR和Western blot方法检测后代血液中外源NA-Mx联合基因的表达情况。结果表明:PCR检测证实外源Mx-NA联合基因已在精子基因组中表达,精液PCR阳性率为33.33%(2/6),且能通过体外受精在后代中检测到,后代PCR检测的阳性率为31.43%(11∕35),Western blot检测F1代阳性率为28.6%(10/35)。以上结果证明外源Mx-NA基因能稳定整合到鸡的基因组中,表明睾丸注射法是一种操作简单、有效的制备抗病转基因鸡的方法。     

精子载体法在转基因动物中的应用与前景

作为动物雄性生殖细胞的精子可与卵子受精而形成受精卵。利用精子的这一特点,以其作为载体将外源基因导入动物体内而制作转基因动物。精子载体法以其简便、有效而引起人们的关注。本文对精子载体法的可行性、研究现状、机理及存在的问题等作一综述。     

雄性生殖细胞介导外源DNA转移的新方法

介绍并评价在传统精子载体法的基础上的改进与发展,即辅以生精细胞显微授精的基因转移;输精管注射法体内转染成熟精子和曲细精管注射法转染生精干细胞.这些方法的应用将为哺乳动物基因转移提供更为方便和高效的技术手段.     

Technical development for production of gene-modified laboratory rats

Transgenic rats have been used as model animals for human diseases and organ transplantation and as animal bioreactors for protein production. In general, transgenic rats are produced by pronuclear microinjection of exogenous DNA. Improvement of post-injection survival has been achieved by micro-vibration of the injection pipette. The promoter region, structural gene, chain length and strand ends of the exogenous DNA are not involved in the production efficiency of transgenic rats. Exogenous DNA prepared at 5 microg/ml seemed to be better integrated than lower and higher concentrations. Intracytoplasmic sperm injection (ICSI) has been successfully achieved in rats using a piezo-driven injection pipette. The ICSI technique has not only been applied to rescue infertile male strains but also to produce transgenic rats. The optimal DNA concentration for the ICSI-tg method (0.1 to 0.5 microg/ml) is lower than that for the conventional pronuclear microinjection. Production efficiency was improved when the membrane structure of the sperm head was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. For successful production of transgenic rats with a modified endogenous gene, establishment of embryonic stem cell lines or alternatively male germline stem cell lines and technical development of somatic cell nuclear transfer are still necessary for this species.     

端粒逆转录酶基因修饰对绵羊骨髓间充质干细胞端粒酶活性影响的研究

骨髓间充质干细胞(BMSC)具有多向分化的特性,但其生命周期有限,为进一步探讨端粒逆转录酶(TERT)对端粒酶活性的影响,本试验利用将外源性TERT导入间充质干细胞技术,构建重组表达载体pcDNA3.1-EGFP-TERT,将其转染BMSC,通过筛选及鉴定,结果显示成功构建了重组表达载体pcDNA3.1-EGFP-TERT.将TERT-BMSCs在体外进行传代培养及生长曲线的测定,利用PCR技术对其原有干细胞因子的表达情况进行了鉴定.结果显示,TERT-BMSC具有快速扩增的能力,细胞形态良好,且仍具有干细胞特性.本试验结果揭示了外源性TERT基因的表达能激活绵羊BMSC的端粒酶活性.     

Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration

1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.     

NON-SURGICAL STERILISATION OF RAMS USING A SCLEROSING AGENT

Injection of 0.25 ml of a solution of 3.6% formaldehyde in 90% ethanol direct into the caudae epididymides caused sterility in the rams treated. No live sperm were seen in ejaculates collected 35, 57, 91 and 196 days post-injection. The rams retained their libido and were mated to ewes between 2 and 3 months post-injection with no pregnancy ensuing. The technique offers a simple, safe, quick and effective method of sterilising rams. Attempts to produce sterilisation by injection of the sclerosing agent into the vas deferens were not successful.     

Multinuclear–Multiflagellar Sperm Defect in a Bull – a New Sterilizing Sperm Defect

The development and use of modern techniques, such as intracytoplasmic sperm injection (ICSI), gene knockout and sperm fluorescence in situ hybridization with chromosome- specific probes, have significantly increased our knowledge about sperm defects. We describe a new oligoasthenoteratozoospermic defect in a bull. Because of its morphological characteristics the defect was named the multinuclear-multiflagellar sperm defect. All spermatozoa in the ejaculate were abnormal. Many of the spermatozoa had multiple nuclei and multiple sperm tails. All spermatozoa lacked an acrosome, and only seldom did spermatozoa have a mitochondrial helix in the midpiece area. Meiosis and spermiogenesis were severely affected in this otherwise phenotypically normal bull. The sperm defects resembled the phenotype of a targeted gene knockout Hrb(-/-) (HIV-1 Rev-binding/interacting protein) mutant mouse strain, which is expressed as sterility in males, while females remain fertile. Since the father of this bull has been extensively used in at least three countries the defective gene has possibly become widespread in the red and white breeds (Ayrshire, Swedish Red and White, Norwegian Red) in the Nordic countries. However, it is not proved that the father of this bull is a carrier of this defect.     

Effects of sperm pretreatment on efficiency of ICSI-mediated gene transfer in pigs

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.     

猪精子结合和内化外源基因的影响因素研究

精子介导基因转移(SMGT)是目前转基因动物研究中简单而高效的方法之一,其中精子结合和内化外源基因的效率是精子介导转基因成功的关键.本试验以DIG标记的线性化EGFP作为示踪基因来检测猪精子结合和内化外源基因的影响因素,结果表明:猪精子能够自发结合外源基因,结合部位主要在精子顶体后区.精子结合外源DNA的阳性率随共孵育时间延长而增加,在37℃或39℃时孵育60 min后,阳性率不再增加;在17℃时孵育90min后,阳性率不再增加.在检查的15只公猪样本中,精子与外源DNA的结合率为6.57%～35.81%,内化率为2.99%～24.66%,个体间差异显著(P<0.01).精浆能够强烈抑制外源基因的结合和内化,脂质体及DMSO能够显著提高转染效率.死精子能够结合外源基因,但不能将其内化;反复冻融导致质膜破裂的精子对外源基因有更高的结合率,且不受个体影响.     

Influence of different methods of collection from the canine epididymides on post-thaw caudal epididymal sperm quality

Canine epididymal sperm was collected from the cauda epididymis using 2 different methods (flushing and mincing) to compare the qualities (the percentage of progressively motile, viable, morphologically abnormal, immature and intact acrosomes) before and after freezing and thawing. No significant difference was noted in the quality of the cauda epididymal sperm immediately after collection and after freezing-thawing between the collection methods, although the mean levels of sperm quality with the flushing method were slightly better than that of the mincing method. The flushing method is simple and free of blood contamination, although the vas deferens was too small to be perfused in only 1 dog, and our results suggest that the flushing method is preferable to the mincing method for collecting sperm from the canine cauda epididymis.