转基因植物中外源基因的整合特性及其研究策略

综述了外源基因在宿主中的整合特性及结构变化的多样性。将外源基因在宿主中的整合方式分为3种：单拷贝单位点整合、串联多联体单位点整合及单拷贝或多拷贝在多位点整合。分析了不同整合方式对表达的影响。详细讨论了植物转基因的整合机制,阐述了以基因组的双链断裂修复为基础的整合模型。最后介绍了国内外的研究者对不同的整合方式所采取的研究策略。     

外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位

为探讨外源基因人基质金属蛋白酶组织抑制物-1（human tissue inhibitor of metalloproteinase-1, hTIMP-1）基因在转基因小鼠家系染色体上的整合和精确定位,应用Southrn印迹检测外源基因在染色体上整合的位点及拷贝数.结果表明,外源基因是以单拷贝、单位点形式整合;应用荧光原位杂交（fluorescence in situ hybridization, FISH）技术检测F4～F20代转基因小鼠中外源基因的整合.结果证明,该家系转基因小鼠自F4代起是纯合子,外源基因整合在17号染色体E区;反向PCR法（Inverse PCR, IPCR）克隆出约3.8 kb外源基因整合位点处的侧翼序列.分析表明,外源基因整合在17号染色体E1.3区,ALK（anaplastic lymphoma kinase, ALK）基因第23个内含子区域.结果提示,获得的转基因小鼠为纯系,外源基因hTIMP-1已稳定整合在转基因小鼠染色体上,并能遗传给后代.     

外源基因在苏云金杆菌染色体上的定点整合及表达

[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法.     

外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展

巴斯德毕赤酵母(P.pastoris)以其独特的生物学和遗传学特征已成功地表达了各种类型的外源蛋白。本文综述了巴斯德毕赤酵母的一些特征和优势,及其产生外源基因多拷贝的原理。了解如何通过多拷贝整合外源基因在巴斯德毕赤酵母中提高外源蛋白表达量、基因拷贝数与表达量的关系,以及如何定量和半定量鉴定拷贝数等,在理论研究和实践应用中,特别是在生物制药工业中具有重要意义.     

外源基因在转基因油菜染色体上的定位及分析

原位杂交技术 ( in situ hybridization,ISH)是基因定位的主要技术之一。近来 ,随着植物细胞染色体制片技术的发展 ,以及酶联放大检测系统的采用 ,在植物中已有低拷贝和单拷贝甚至小于 1 kb的 DNA序列定位的成功报道 [1 ,2 ]。染色体原位杂交技术不仅可以用于基因的物理作图 ,而且可以用来对转基因植物中的外源基因进行染色体定位 [3 5] 。研究表明 ,外源目的基因在转基因植物中的表达与整合位点有关 [6] 。因而 ,进行外源基因在转基因植物染色体上的定位以及研究外源基因的整合位点与表达之间的关系 ,对于开发和利用转基因植物具有重要…     

转基因动物中基因转移和表达的分子机制

本综述着重讨论在转基因动物中外源基因转移及表达的分子机制,包括外源基因在宿主染色体上整合的分子基础及其影响因素：启动子、内含子、增强子、反式作用因子及载体等各种因素对外源基因在宿主内表达的调节。     

植物转基因的失活与沉默

植物外源基因转化研究揭示有相当比例的植物转基因表现失活与沉默。这一现象涉及转基因重复拷贝之间的异源配对,基因顺序的甲基化,转录后的衰退调控以及插入位点染色质高级结构的改变,并与外源基因的随机整合有关。克服转基因失活的方法包括单拷贝转基因子代筛选,构建细胞核基质支架附着区载体以及采用具有特殊功能的启动子与增强子。转基因失活问题的发生提示人们,外源基因的转化与整合存在一些特有的遗传机制。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k2tPA)、扩增基因(dhfr)、重组酶识别序列(FRT)及筛选基因(neo),且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点(Hotspot)区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统(FLP-FRT)将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17.1μg/106cell.24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

反转录病毒基因转移载体

80年代初期,以反转录病毒作载体进行基因转移的技术迅速发展起来。以反转录病毒作载体进行基因转移具有以下几个方面的特点:宿主细胞范围广;病毒感染滴度高;病毒感染宿主细胞后,细胞不发生病变,可以稳定地表达外源基因;基因转移效率高,病毒基因往往以低拷贝整合,易于外源基因表达的研究;反转录病毒较小,易于操作,容纳的外源基因较大(10kb左右);基因转移后不发生重排;经特殊构建的反转录病毒载体,不易产生感染性病毒粒子,比较安全。为了构建一个基因转移效率高、宿主范围广、安全的反转录病毒基因转     

植物转基因的失活与沉默

植物外源基因转化研究揭示有相当比例的植物转基因表现失活与沉默。这一现象涉及转基因重复拷贝之间的异源配对,基因顺序的甲基化,转录后的衰退调控以及插入位点染色质高级结构的改变,并与外源基因的随机整合有关。克服转基因失活的方法包括单拷贝转基因子代筛选,构建细胞核基质支架附着区栽体以及采用具有特殊功能的启动子与增强子。转基因失活问题的发生提示人们．外源基因的转化与整合存在一些特有的遗传机制。     

Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F(1) plants were self-pollinated and the F(2) generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F(2) plants with transposed Ds-bar was 100% stable, whereas only 23% of F(2) plants carrying Ds-bar at the original site expressed the transgene product stably. In F(3) and F(4) populations, transgene expression in 81.5% of plants from progeny of F(2) plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.     

Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase

Cre is a 38-kD protein from bacteriophage P1 that catalyzes site-specific recombination between 34-bp loxP sequences. Our previous work has shown that Cre can perform site-specific excisive recombination not only in prokaryotes, but also in eukaryotes such as yeast and cultured mammalian cells. In this work we show that intermolecular Cre-mediated recombination can specifically direct the integration of a loxP-containing circular DNA into a chromosomal loxP site, both in yeast and in mammalian cells. The resulting integrants are predominantly simple single-copy insertions. Cre-mediated recombination thus provides a simple way to direct single-copy site-specific integration of exogenous DNA into the eukaryotic genome.     

Chromosomal complementation using tn7 transposon vectors in enterobacteriaceae

Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn7 transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn7 systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn7 and transposase vectors to provide a more versatile system. Transposition of Tn7 at the chromosomal attTn7 site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn7 vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.     

转pGH基因猪外源基因染色体定位研究

应用地高辛标记探针对转pGH基因(猪生长激素基因)猪染色体进行原位杂交,经胶体金抗体、银增强放大系统检测外源pGH基因在染色体上的整合位点。研究表明,转基因阳性个体间外源基因整合位点存在差异,但对个体而言,外源基因总是集中分布于某一特定的染色体上。本研究将为探究外源基因整合位点与其表达效率的关系及今后定点整合的研究提供理论指导。     

Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization

 Using fluorescence in situ hybridization (FISH) we localized introduced genes on metaphase chromosomes of barley, wheat, and triticale transformed by microprojectile bombardment of microspores and scutellar tissue with the pDB1 plasmid containing the uidA and bar genes. Thirteen integration sites were detected in the nine lines analysed. Southern analysis showed that three or more copies of the plasmid were present in the lines. In a triticale line containing four copies three different integration sites were identified, indicating that the method described is sensitive enough for the detection of single-copy integrations. There was a slight tendency towards the localization of transgenes in distal chromosome regions. Using the GAA-satellite sequence for chromosome banding, the chromosomes containing the inserted genes were identified in most cases. Two barley lines derived from the same transformant showed a totally different integration pattern. Southern analysis confirmed that the inserted genes were segregating independently, resulting in different integration patterns among the progeny lines. The application of the FISH technique for the analysis of transgenic plants is discussed. Received: 28 October 1996/Accepted: 15 November 1996     

pLR: A lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site) - reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.     

Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.     

人乳铁蛋白(hLF)转基因山羊的遗传背景分析

以随机整合方式获得的转基因动物外源基因的拷贝数、整合位点及染色体核型等遗传背景并不清楚,可能会存在外源基因的沉默整合、无效整合、毒性整合以及其表达水平不可预测等问题。文中选取了6只原代(F0)及其相对应的子一代(F1)的人乳铁蛋白(hLF)转基因山羊作为研究对象,分别颈静脉采血、提取DNA,通过染色体核型分析、实时荧光定量PCR(qPCR)、ELISA和Westernblotting等检测技术,研究其外源基因的遗传背景与表达水平。结果显示,6只F0代转基因山羊的染色体没有明显的形态变异、数量改变等异常情况。相对拷贝数高低不同(2–16),且能够稳定地遗传给下一代,F0和F1代hLF基因拷贝数一致。F1代转基因山羊表达hLF水平最高可达1.12 g/L(L3-1,拷贝数8)。结果表明,整合的外源基因能够稳定地遗传下一代,也没有对转基因山羊个体的生长发育造成障碍,而且拷贝数高低与hLF表达水平无明显的相关性,这为转基因山羊及其他转基因动物的新品种培育奠定了基础,解析了遗传背景。