慢病毒载体法制备转基因动物研究进展

慢病毒能够感染分裂细胞和非分裂细胞,因而被发展成为重要的转基因载体,已成为制备转基因动物的一种工具,转基因效率明显提高。该文介绍了制备转基因动物的技术方法,比较了慢病毒载体制备转基因动物的特点和优势,介绍了慢病毒载体安全设计的发展,并将近年来国内外利用慢病毒载体法制备转基因动物的研究进行了概述。     

精子载体法制作转基因动物的可行性

精子载体法转基因由于具有简便易行、对卵原核无损伤等特点,而成为最具诱惑力的转基因方法之一近年来,已经通过该方法获得了多种转基因动物,从而对精子载体法制作转基因动物的可行性提供了证据.笔者对精子载体法的可行性、机制、提高转基因效率等方面作了简要综述.     

精子载体法制作转基因动物的可行性

精子载体法转基因由于具有简便易行、对卵原核无损伤等特点，而成为最具诱惑力的转基因方法之一。近年来，已经通过该方法获得了多种转基因动物，从而对精子载体法制作转基因动物的可行性提供了证据。笔者对精子载体法的可行性、机制、提高转基因效率等方面作了简要综述。     

精子载体转基因的研究进展

精子载体法培养转基因动物是目前操作最简便，成本低廉且危险隐患相对较低的一种转基因方法。近10年来的研究结果表明，精子载体法可得到转基因阳性动物，并且引起了更多科研人员的关注。     

转基因技术及转基因动物研究进展

转基因技术为从分子到个体多方面研究基因提供了新的方法和思路。本文介绍了原核注射法，载体法等转基因技术和转基因动物的检测方法。以及转基因动物在提高动物生长速度，提高动物产毛性能，制药，抗病育种以及由于转基因动物所产生的一些问题进行了探讨。     

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.     

转基因动物制作技术及其基因载体研究进展

论文概述了家畜转基因动物的制作方法,初步分析了用于转基因技术的基因载体种类,深入探讨了不同载体的优缺点,并展望了基因载体的发展趋势。     

转基因动物制作技术及其基因载体研究进展

论文概述了家畜转基因动物的制作方法,初步分析了用于转基因技术的基因载体种类,深入探讨了不同载体的优缺点,并展望了基因载体的发展趋势.     

转基因动物研究进展

本文讨论了ＤＮＡ显微注射法、载体法、细胞培养法和胚泡细胞注入法等产生转基因动物的方法,以Ｓｏｕｔｈｅｒｎ印迹为主,包括ＰＣＲ阳性产物测序法等转基因动物检测方法;乳腺表达系统和基质结合区（ＭＡＲ）等转基因动物表达系统以及转基因动物在分子生物学、畜牧兽医以及医学等领域中的应用。     

利用动物乳腺生物反应器生产人凝血因子IX

转基因动物乳腺生物反应器是利用动物乳腺特异性启动子调控元件指导外源基因在乳腺中特异性表达,并从转基因动物奶中获取重组蛋白.利用转基因动物乳腺生物反应器生产人凝血因子Ⅸ是一种新型的生物制药方法且具有广阔的应用前景.本文就转基因动物乳腺生物反应器生产人凝血因子Ⅸ的载体构建、研究现状以及存在的问题等作一综述.     

Design of vectors for transgene expression: The use of genomic comparative approaches

The design of transgenes has always been limited by the extent of available information on the endogenous locus whose expression pattern had to be replicated. Those genes whose expression domain had not been entirely documented resulted, usually, in transgenes with an unpredictable expression patterns and suboptimal performance in transgenic animals. The use of genomic comparative approaches, highlighting evolutionary conserved homologous DNA sequences, helps to identify crucial regulatory elements that are associated to a given expression domain. The inclusion of these conserved regulatory sequences in transgenic constructs would normally result in optimal expression levels of transgenes in recipient animals. The use of artificial chromosome-type transgenes usually ensures the inclusion of these preserved regulatory elements that are required for the faithful expression of the gene. These constructs could also contain insulators, a subset of regulatory sequences whose application is being addressed in transgenesis. Therefore, the generation of transgenic animals with genomic-type constructs is the recommended approach to achieve optimal transgene expression, according to the expected pattern of the corresponding endogenous locus.     

Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control

This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastric animals offers a promising biological approach to relieve the requirement for dietary phosphate supplements and to reduce phosphorus pollution from animal agriculture.     

Transgenic mouse offspring generated by ROSI

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.     

CRISPR/Cas9 knock-in介导的输卵管特异性表达转基因鸡研究进展

利用动物生物反应器生产重组蛋白是一种具有应用前景的生物技术。鸡输卵管生物反应器是理想的动物生物反应器之一,其优点在于表达的外源蛋白能够分泌到蛋清中,可避免蛋白提取过程中对鸡本身造成伤害,同时蛋清成分简单,便于后期的纯化。目前利用慢病毒结合原始生殖细胞(PGCs)制备转基因鸡被认为是最可行的方法,但因外源基因随机整合且生殖系传递效率较低,使转基因鸡研究受到技术上的限制。而2013年问世的CRISPR/Cas9基因敲入(CRISPR/Cas9 knock-in)技术能够使外源基因精准定向插入基因组特异性位点,这对生产输卵管特异性转基因鸡具有重大意义。文章综述了鸡输卵管反应器的研究进展、CRISPR/Cas9 knock-in技术在输卵管特异性表达转基因鸡研究和鸡育种领域的应用现状,并指出了目前存在的问题和相应的解决办法。     

稳定转化家蚕BmN细胞表达人粒细胞-巨噬细胞集落刺激因子

为了建立稳定转化家蚕细胞持续表达外源基因的技术体系,构建了基于piggyBac转座子的带有人粒细胞-巨噬细胞集落刺激因子基因(hGM-CSF)和新霉素抗性基因(neo)表达元件的转基因载体pigA3GFP-IE-neo-FH-hGM-CSF-polyA-fib-L-intron1,以及带有hGM-CSF和吉欧霉素(zeocin)抗性基因的昆虫细胞转化载体pIZT-IE-hGM-CSF,分别转染家蚕卵巢BmN细胞,并以含相应抗生素G418或zeocin的培养液筛选,得到稳定的转化细胞系FH-hGM-CSF和IE-hGM-CSF。ELISA检测结果显示,hGM-CSF在FH-hGM-CSF和IE-hGM-CSF转化细胞系的表达水平分别为1.534 55 fg/个细胞和2.227 38 fg/个细胞。     

Use of Transgenic Animals to Improve Human Health and Animal Production

Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed.     

AMV和WCMV在转基因红三叶中的基因累集

随着转基因技术的发展及其在植物育种中的应用，出现了一大批表现优良、经济效益高的转基因植物，但生产实践要求一种植物同时具有多个优良的转基因性状。目前解决这一问题的方法主要是基因累集(Gene pyramiding)，就是将多个基因集中到同一植株，可以将已经转入1个基因的植株做原材料，用农杆菌介导或基因枪等方法将另一基因转进去。也可以采用人工杂交，在不同转基因植株之间进行人工授粉，从后代中筛选出所需要的基因型。本研究对抗苜蓿花叶病毒(AMV)的转基因红三叶和抗白三叶花叶病毒(WCMV)的转基因红三叶进行了人工杂交，以期获得兼有2个基因、同时抵抗2种病毒的新植株，为下一步的育种打好基础。几乎所有的杂交组合都结了种子，杂交一代的发芽率随杂交亲本转基因拷贝数的不同而有所变化，只有1个转基因拷贝的亲本其杂种发芽率最高。对所有幼苗先用PCR进行检测，从中挑出较理想的基因型作进一步的分子生物学分析。Southern和Northern印迹分析结果表明，有些杂交一代的转入基因多于1个拷贝，有一部分同时兼有2种抗病基因，并且得到了表达。为进一步检测其抗病性，对它们进行了温室接种和田间评估，结果显示，尽管有的个体Northern分析呈阳性，但在田间仍然对病毒敏感，不过其感病程度都比对照有显著降低。大多数兼有2种转入基因的植株都能同时抵抗2种病毒，可以作为免疫个体供下一步育种之用。与遗传转化相比，人工杂交以其操作简单、省时省力、预见性强和准确率高而具有明显优势，是常规育种方法在分子育种中的具体体现。