植物基因打靶研究现状

基因打靶是八十年代后期发展起来的一门新兴的遗传工程技术,基因打靶技术在探索生物的基因功能,消除转基因的沉默,提高转基因的稳定性和表达效率以及基因治疗等方面均取得了进展。基因打靶技术的应用前景广阔,它的产生是遗传工程领域的一次革命。目前,基因打靶在植物上的应用研究才刚起步。本文针对基因打靶在植物上的研究现状作一综述。     

植物基因打靶研究现状

基因打靶是八十年代后期发展起来的一门新兴的遗传工程技术,基因打靶在探索生物的基因功能,消除转基因的沉默,提高转基因的稳定性和表达效率以及基因治疗等方面的均取得了进展,基因打靶技术的前景广阔,它的产生是遗传工程领域的一次革命。目前,基因打靶在植物上的应用研究才刚起步。本文针对基因打靶在植物上的研究现状作一综述。     

条件基因打靶技术在组织稳态研究中的应用

基因打靶技术作为最有效的定向修饰小鼠基因组的技术手段在揭示基因的生理功能、研究人类疾病的遗传机制以及寻找新的药物靶标的过程中发挥重要的作用.基于Cre-LoxP定位重组系统的条件基因打靶技术的发展使得基因失活可以限制在特定发育阶段的特定组织或细胞内,这一优势使得条件基因打靶技术成为解析哺乳动物基因功能的主要研究手段.本文将主要介绍军事医学科学院在建立小鼠条件基因打靶技术平台,以及应用这一技术研究TGF-β/Smad等重要信号通路在维持组织稳态和抑制疾病的生理功能和机制方面的主要进展.     

基因打靶技术在现代生物学研究中的应用

尽管基因打靶技术的先驱者2007年才获得诺贝尔生物学或医学奖,基因打靶技术在现代生物学研究中的应用却已经有25年的时间.基因打靶技术的发明和应用革命性地改变了现代生物医学研究的面貌,催生了许多生物医学和药物研发领域的前沿研究,并直接导致了生物医学研究领域中的许多突破性进展.基因打靶技术的发明使得科学家第一次能对关于特定基因生理功能的假设进行实验验证,并通过基因打靶研究真正建立基因和疾病间的因果关系.近年来,基因打靶技术在现代生物学研究中的应用日益深入和广泛.     

基因打靶技术:开启遗传学新纪元

基因打靶技术作为最有效的定向修饰小鼠基因的技术手段在揭示基因的生理功能、研究人类疾病的遗传机制以及寻找新的药物靶标的过程中发挥着重要的作用。近年来, 随着条件基因打靶技术的发展使基因失活可以限制在特定时段特定组织或细胞内。文章将主要介绍基因打靶技术的发展简史、近期进展以及在其他模式动物中的应用。     

体细胞基因打靶制备动物乳腺生物反应器的策略与应用

在转基因动物研究中,由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”,致使转基因动物外源基因的表达水平不高并且差异较大。为此,利用定位整合优势,对以基因同源重组为基础的基因打靶技术进行了大量研究。介绍了就利用体细胞基因打靶和核移植技术制备动物乳腺生物反应器的策略和应用情况做一综述,并对提高基因打靶效率的各种策略,打靶细胞的选择,转基因细胞核移植的低融合事件以及基因打靶制备乳腺生物反应器的优越性进行分析。     

植物基因打靶技术及其应用

本文对植物基因打靶技术的原理、操作程序、打靶效率的影响因素及其在植物中的应用现状进行了综述,并就如何有效的提高打靶效率提出了建议,同时对该技术在植物学研究领域中的应用前景进行了展望。     

体细胞基因打靶-核移植技术研究进展

转基因效率与外源基因表达水平低的现状一直是制约动物生物反应器研究与产业化的主要技术瓶颈。体细胞克隆动物的成功和胚胎干细胞基因打靶技术的逐步完善使得体细胞基因打靶与核移植技术的结合使用成为可能,这就为生产遗传修饰家畜提供了一种新的手段,为动物生物反应器的成功研制提供了新的技术途径。从体细胞基因打靶的载体设计、转染系统的建立、中靶细胞的筛选和鉴定以及培养体细胞寿命等方面阐述了体细胞基因打靶—核移植技术体系的最新研究进展,并对其在异种器官移植、建立动物疾病模型、提高家畜生长性能以及生产药用蛋白等各个领域中的应用前景作了展望 。     

腺相关病毒载体介导的基因打靶技术的研究进展

基因打靶技术在基因治疗和基因功能研究方面都有重要作用,而基因导入系统的有效性是影响中靶效率的重要因素.AAV(腺相关病毒)载体介导的基因打靶在保证有效转移DNA的同时,还具有非致病性、宿主范围广、高中靶效率和高保真性等诸多优点,使AAV成为目前人类基因治疗研究中最理想的病毒载体之一.本文就近几年AAV介导基因打靶的研究进展作一综述.     

基于体细胞克隆的猪基因打靶技术研究进展

基因打靶猪在农业和生物医药领域均有广泛用途。由于猪的能参与生殖系嵌合体的多能性干细胞尚未建立成功,基因打靶猪的培育主要是通过体细胞克隆技术来实现。最初,人们在体细胞上通过传统的同源重组技术成功地建立了基因敲除克隆猪,但体细胞在体外的增殖能力有限,传统同源重组在体细胞的打靶效率极低。虽经十多年的发展,但在世界范围内获得的基因打靶猪屈指可数。最近,三种工程核酸酶介导的基因编辑技术(ZFN、TALEN和CRISPR/Cas9)的出现,使体细胞的基因打靶效率大大提高。多个实验室将其用于猪,实现了高效基因打靶,并在很短的一段时间里,获得了一系列基因打靶猪。就传统同源重组技术以及近几年发展起来的新兴基因编辑技术在基因修饰猪的应用研究进展进行了综述。     

基因打靶技术的研究进展

基因打靶技术是一项新兴的分子生物学技术,是利用外源DNA与受体细胞染色体DNA上的同源序列之间发生重组,并整合在预定位点上,从而改变细胞遗传特性的方法。它的产生是遗传工程领域的一次革命,为发育生物学、分子遗传学、免疫学及医学等学科提供了一个全新的、强有力的研究手段。目前基因打靶技术在研究基因的结构和功能、表达与调控,转基因及基因治疗等方面均取得了进展。但基因打靶技术仍存在一些问题,主要是打靶的效率太低。本文综述了基因打靶技术的原理、操作程序并对提高基因打靶效率的可能途径进行了探讨。 Progress on Gene Targeting LIU Hong-quan1,DAI Ji-xun1,YU Wen-gong2,YANG Kun-feng1 1.Ocean University of Qingdao,College of Marine Life Sciences,Qingdao 266003,China; 2.Institute of Marine Drugs and Foods,Qingdao 266003,China Abstract:Gene targeting is a rising technology in molecular biology,which is defined as the introduction of exogeneous DNA to specific site in genome by homologous recombination,and consequently change the hereditary character of the cell.This technology provides a new and powerful means for research in developmental biology,molecular genetics,immunology and medicine.Progresses have been made in exploring gene structure and function,gene expression and regulation,transgene and gene therapy with the application of gene targeting.But there are some problems in gene targeting,especially for the low efficiency.This article just provided a review of the principle and program of gene targeting,and discussed the possible approaches to increase the efficiency of gene targeting. Key words：gene targeting;homologous recombination;targeting vector;targeting efficiency     

基因打靶技术及其应用前景

基因打靶是近年来发展起来的对细胞基因组中的某一基因进行定点操作的生物技术。综述了基因打靶的筛选系统,影响基因打靶的几个主要因素及其解决方法,总结了基因打靶在各个学科领域中的应用。     

A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.     

Gene targeting using zinc finger nucleases

The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by introducing DNA double-strand breaks in target genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.     

A review of current large‐scale mouse knockout efforts

After the successful completion of the human genome project (HGP), biological research in the postgenome era urgently needs an efficient approach for functional analysis of genes. Utilization of knockout mouse models has been powerful for elucidating the function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and gene targeting are two independent techniques for making knockout mice from embryonic stem (ES) cells. Gene trapping is high‐throughput, random, and sequence‐tagged while gene targeting enables the knockout of specific genes. It has been about 20 years since the first gene targeting and gene trapping mice were generated. In recent years, new tools have emerged for both gene targeting and gene trapping, and organizations have been formed to knock out genes in the mouse genome using either of the two methods. The knockout mouse project (KOMP) and the international gene trap consortium (IGTC) were initiated to create convenient resources for scientific research worldwide and knock out all the mouse genes. Organizers of KOMP regard it as important as the HGP. Gene targeting methods have changed from conventional gene targeting to high‐throughput conditional gene targeting. The combined advantages of trapping and targeting elements are improving the gene trapping spectrum and gene targeting efficiency. As a newly‐developed insertional mutation system, transposons have some advantages over retrovirus in trapping genes. Emergence of the international knockout mouse consortium (IKMP) is the beginning of a global collaboration to systematically knock out all the genes in the mouse genome for functional genomic research. genesis 48:73–85, 2010. © 2010 Wiley‐Liss, Inc.     

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines.     

Using nuclear targeting signals to enhance non-viral gene transfer

Summary Gene therapy involves the introduction of DNA-encoding therapeutic gene products into appropriate cells of an affected individual. The limitations of the approach relate largely to the poor efficiency of the delivery of the therapeutic DNA to the nucleus. This review examines recent work in the area of non-viral gene transfer, building on developments in the field of nuclear protein import and their application in the field of non-viral gene transfer. In particular, advances in the area of enhancing DNA targeting to the nucleus are discussed, including the use of modular nuclear targeting signals recognised by the cellular nuclear import machinery and DNA condensing agents to facilitate passage through the nuclear pore. Optimising nuclear DNA delivery through these and other strategies should assist greatly in rendering gene therapy a viable and realistic possibility for treating disease.     

Aptamer-guided gene targeting in yeast and human cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.     

The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination

Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.