用慢病毒载体制备转基因动物的研究进展

慢病毒是逆转录病毒的一种 ,具有逆转录病毒的基本结构 ,但也有不同于逆转录病毒的组份和特性 ,作为基因治疗载体发展起来 ,最近已用于转基因动物制备。慢病毒像其它逆转录病毒一样 ,其基因组经逆转录后能整合在宿主DNA上 ;由于病毒载体经改构后 ,不在宿主细胞增殖 ,不会导致寄主细胞的死亡 ,被它感染的或转化的动物细胞能够连续传代 ;这种载体的最大优势是能感染静止细胞和不产生嵌合体动物。详细介绍了慢病毒载体构建原理、近年慢病毒载体在转基因动物制备方法和应用研究的新进展。     

人c-myc转基因细胞的建立

目的:建立人c-myc转基因细胞。方法:通过成功构建c-myc逆转录病毒表达载体,并经脂质体介导转染包装细胞293T,收集产重组病毒的293T培养上清,运用NIH3T3细胞测定了病毒滴度,用适当浓度的病毒感染L929细胞,经用Zeocin选择性培养基筛选细胞。结果:得到稳定高表达c-myc基因的L929转基因细胞。结论:运用逆转录病毒转染法可得到高表达的转基因细胞。     

携带猪β干扰素基因的鸡输卵管生物反应器逆转录病毒载体的构建

为构建携带猪β干扰素基因的鸡输卵管生物反应器逆转录病毒载体,用PCR技术扩增了猪β干扰素基因和卵清蛋白5调控序列,然后将其重组到切除了P70启动子的逆转录病毒载体pLNHX中。用酶切和PCR等方法对重组质粒进行鉴定,结果表明,卵清蛋白5‘调控序列和猪β干扰素基因已正确克隆至逆转录病毒载体pLNHX中。为进一步制备高滴度,高感染性的逆转录病毒,从而制作鸡的输卵管生物反应器奠定实验基础。     

逆转录病毒介导myostatin在鸡胚胎中的过表达与检测

鸡的胚胎已经成为发育生物学、免疫学和癌症等学科的重要实验模型之一。以鸡胚胎为实验模型,以鸡myostatin为靶基因,利用逆转录病毒介导和原位杂交技术相结合的方法来研究目的基因的鸡胚胎转导和检测分析。实验结果表明：逆转录病毒介导的目的基因myostatin能够有效整合到胚胎基因组中,且具有转录活性;胚胎原位杂交检测能够准确直观的反映病毒的整合部位和转入基因的表达水平。不但为进一步研究鸡myostatin基因在胚胎发育中的功能奠定基础,同时在方法上也进行了简单的优化尝试。     

转基因鸡的研究策略

转基因研究工作开展十几年来,成就斐然。转基因鸡的研究策略论述了生产转基因鸡的几种方法和鸡的基因组操作研究进展,并详细地阐述了转基因鸡胚胎的体外培养技术。指出：转基因鸡投入生产以前必须查明其表型改变对生态平衡的影响。     

转基因鸡的制备方法探讨

由于鸡本身的众多优点,转基因鸡的研究逐渐受到了研究者的重视,简要介绍了转基因鸡的发展史,对各种转基因技术方法进行了比较和分析,提出了每种方法的优缺点,尤其在精子介导法上作了较为详细的论述,并展望了转基因鸡的应用前景。     

转基因RNAi技术在动物抗病毒育种中的应用

病毒对动物和人类健康都是极大的威胁,抗病毒疫苗虽然能在一定程度上预防病毒病,但目前几乎还不能对变异的传染病进行抗病毒治疗。尽管RNA干扰研究到目前才短短十几年,其作用机理已基本清楚。RNAi能够非常有效的抑制病毒体内复制,其介导的抗病转基因动物的研究相继取得了阶段性进展,抗疯牛病转基因羊和牛,抗内源性逆转录病毒猪以及抗核型多角体病毒病的转基因家蚕已经成功获得。尽管如此,目前的研究主要还是集中在细胞水平及小鼠模型方面,获得的转基因动物种类和数量有限,但为培育动物抗病毒品种提供了理论依据和技术支撑。随着转基因技术的不断的进步和成熟, RNA干扰技术将成为动物抗病毒育种中最有应用前景的方法之一。     

转基因鸡研究现状及其展望

自1987年美国农业部地区家禽研究所(USDA,ARS,Regional Poultry Research Laboratory)成功地得到转基因鸡以来,有关研究已引起学术界和产业界的浓厚兴趣。今年6月25日至6月28日在美国康乃尔大学召开的第二届动物遗传工程国际学术讨论会上提交大会交流的20篇论文中,转基因鸡及有关研究就占了6篇。与其他转基因动物的研究相比较,转基因鸡的研究有其重要意义。     

逆转录病毒表达系统及其在外源蛋白高效表达中的应用

逆转录病毒表达系统是一种新的重组蛋白高效表达系统,它由逆转录病毒载体,包膜蛋白载体和包装细胞系构成,在基因治疗和生物制药领域都有很大的应用潜力。逆转录病毒和宿主细胞基因组的重组倾向于发生在转录活性区;口炎疱疹病毒-G蛋白 (vesicular stomatitis virus G, VSV-G)能有效扩大逆转录病毒感染宿主细胞的范围、提高逆转录病毒的感染效率;用高滴度的重组病毒感染细胞,经过简单的筛选即可获得高表达细胞株。本文对逆转录病毒表达系统的组成、感染的特点和机制及其应用前景作了概述。     

转基因鸡的研究策略

论述了禽类转基因的特殊性、生产转基因鸡的几种方法及存在的问题,指出转基因鸡的研究具有巨大潜力。     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.     

In vivo cooperation of two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, leads to transformation and immortalization of chicken myelomonocytic cells.

To investigate a possible in vivo cooperation between the p61/63myc and P135gag-myb-ets proteins, we used a previously constructed retrovirus, named MHE226, which contains the fused v-myb and v-ets oncogenes of the E26 retrovirus and the v-myc oncogene of MH2. For that purpose, chicken neuroretina cells producing MHE226 and pseudotyped with the Rous associated virus-1 (RAV-1) helper virus were injected in 1-day-old chickens. In control experiments, we also injected chicken neuroretina cells producing E26 (RAV-1), RAV-1 alone, or constructs lacking one of the oncogenes of MHE226. The average life span of MHE226-infected chickens is half that of E26-infected chickens. MHE226-infected chickens harbor tumors scattered in many organs, but compared with E26, MHE226 induced a weak leukemia. Study of integration sites suggests that the majority of the tumors results from clonal or oligoclonal events. Cell cultures were derived from the tumors of MHE226-infected chickens and grown in standard medium without addition of exogenous chicken myelomonocytic growth factor. These cells still divide at high rate after more than 100 passages and can thus be considered immortalized. By using several criteria, these cells were characterized as precursors of the myelomonocytic lineages.     

动物转基因研究中的技术方法

转基因动物研究的深入为生物学的发展起到了巨大的推动作用。转基因新技术的不断出现,推动了转基因动物的发展。介绍了几种哺乳动物转基因研究中常见的方法,包括原核显微注射法、逆转录病毒感染法、胚胎干细胞介导法、生殖细胞介导法、基因打靶以及新的比较有应用前景的方法,如PiggyBac、iPS,锌指核酸酶法,并且分别对每种方法的优缺点进行了总结归纳。     

Production of transgenic chickens expressing a tetracycline-inducible GFP gene

There is much interest in using farm animals as ‘bioreactors’ to produce large quantities of biopharmaceuticals. However, uncontrolled constitutive expression of foreign genes have been known to cause serious physiological disturbances in transgenic animals. The objective of this study was to test the feasibility of the controllable expression of an exogenous gene in the chicken. A retrovirus vector was designed to express GFP (green fluorescent protein) and rtTA (reverse tetracycline-controlled transactivator) under the control of the tetracycline-inducible promoter and the PGK (phosphoglycerate kinase) promoter, respectively. G0 founder chickens were produced by infecting the blastoderm of freshly laid eggs with concentrated retrovirus vector. Feeding the chickens obtained with doxycycline, a tetracycline derivative, resulted in emission of green body color under fluorescent light, and no apparent significant physiological dysfunctions. Successful germline transmission of the exogenous gene was also confirmed. Expression of the GFP gene reverted to the pre-induction levels when doxycycline was removed from the diet. The results showed that a tetracycline-inducible expression system in transgenic animals might be a promising solution to minimize physiological disturbances caused by the transgene.     

鸡囊胚细胞嵌合体制作技术研究及其应用前景

家鸡X期囊胚细胞(BCs)嵌合体技术,既是利用转基因技术进行家鸡品种改良和凭借转基因家鸡生物反应器生产医用蛋白等研究领域的关键技术,也是利用BCs冻存家鸡和珍稀鸟类双亲种质资源实现鸟类品种资源多样性保护、利用和挽救珍稀濒危鸟类的重要途径。从家鸡BCs嵌合体制作技术的基本过程：(1) 羽色嵌合体家鸡模型的建立;(2) 囊胚的分离与消化;(3) 受体种蛋的致弱处理;(4) 受体种蛋的开窗（包括部位、方法及封口技术等）;(5) 供体细胞导入受体胚（显微注射或简易操作）;(6) 孵化（常规方法或换壳培养）等几个方面的研究进展、目前存在的问题以及研究方向等进行了系统阐述。Abstract: The technology of producing chicken chimeras using blastodermal cells is very important not only in the field of transgenic chicken bioreactor but also in searching for efficient ways to conserve avian genetic resource. The basic processes for producing chicken chimeras consist of: (1) Setting up the color model; (2) Separating and dissociating of donor embryos; (3) Compromising of the recipient embryos; (4) Windowing and recovering the recipient eggs; (5) Cells injecting; (6) Method of hatching. The progress, obstacles and prospects of producing chicken chimeras via BCs were discussed in this paper.     

Production of transgenic chickens constitutively expressing human erythropoietin (hEPO): Problems with uncontrollable overexpression of <Emphasis Type="Italic">hEPO</Emphasis> gene

The chicken is a promising candidate as a bioreactor for the economical mass production of human therapeutic proteins. Here, we report the successful generation of transgenic chickens that produce high concentrations of human erythropoietin (hEPO) in the blood. Using a Moloney murine leukemia virus (MoMLV)-based pseudotyped retrovirus vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G), the hEPO gene under the control of cytomegalovirus (CMV) promoter was introduced to the blastoderm of freshly laid chicken eggs (stage X). Out of 200 injected eggs, 12 chicks were hatched after 21 days of incubation, and all of the G₀ hatched chicks expressed the vector-encoded hEPO gene. One of the G₀ roosters successfully transmitted the hEPO gene to its G₁ progeny by crossing with non-transgenic hens. The concentration of hEPO protein in the chicken blood serum was as high as 90 μg/mL. Although humans and chickens belong to different classes of the phylogenetic tree, human EPO caused devastating problems in transgenic chickens, including sudden death, polycythemia, vasodilation, and so on, which may be due to the uncontrolled constitutive expression of exogenous protein in the chicken body. Despite many disorders, however, we were able to generate chicks of G₂ generation sired by a rooster of G₁ generation confirming successful establishment of a new line of transgenic chicken characterized by high expression of the hEPO gene. With these chickens, we believe that studies on the evaluating the possibilities of the transgenic animal-mediated bio-pharming and on the hEPO-induced physiological side effects will be greatly facilitated.     

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.