锌指核酸酶技术在动物转基因中的应用

锌指核酸酶(Zinc finger nuclease,ZFN)具有特异性识别并敲除DNA片段中特定基因的特点,通过对DNA片段上的特定基因进行靶向修饰产生新型细胞,是能够应用于动物转基因上的一种新技术.该技术已在一些动物的研究上取得了一定的成果,相对于传统的转基因技术,该技术在简化操作程序、缩短操作时间及提升成功率上都有很大的提高.就常用的动物转基因方法、锌指核酸酶技术的作用原理以及在动物转基因上的应用及其前景进行了论述.     

锌指核酸酶技术在动物转基因研究中的应用

锌指蛋白核酸酶(Zinc Finger Nuclease,ZFN)是一种人工合成酶,含有锌指蛋白和FokⅠ核酸内切酶的剪切结构域,因其能特异性识别并切割DNA序列及其可设计性而被用基因定点突变和外源基因定点整合。这项技术已应用于转基因动物研究中。本文综述了锌指核酸酶技术在转基因研究中的应用进展,并对锌指核酸酶技术的应用前景进行了展望。     

转基因和基因编辑猪的研究进展

猪是重要的农业经济动物,其在遗传背景、解剖学、生理病理学、营养代谢和疾病特征等方面与人类高度相似,是农业生产性状改良、人类疾病动物模型和异种器官移植研究的重要对象。随着ZFN、TALEN和CRISPR等基因编辑技术的出现,转基因或基因编辑猪的研究取得快速发展。本文将结合当前我国转基因及基因编辑猪的研究现状,对基因编辑技术在农业生产性状改良如提高产肉量、改善肉质、提高抗病能力和建立人类疾病模型、动物生物反应器、异种器官移植等方面进行综述,以期为猪遗传改良和医学研究提供参考。     

动物转基因新技术研究进展

 动物转基因技术是将外源基因导入动物体内,外源基因随机整合或定点整合（打靶）在染色体基因组上并得到表达和遗传的生物技术。该技术自发现以来,在畜牧业、医药产业、环境保护以及新型生物材料等领域已得到了广泛应用。本文主要就近年来迅速发展起来的慢病毒载体导入法、精原干细胞法、锌指核酸酶介导的基因打靶、RNA干扰介导的基因沉默等新的高效转基因技术进行了概述。     

胞质内单精子注射制备转基因猪技术的优势与前景

综述了20多年来转基因猪的研究概况,探讨了各种转基因技术的特点,分析了胞质内单精子注射制备转基因猪技术的特点和优势,并展望了单精子注射与人工染色体载体技术相结合制备大片段转基因猪的应用前景。     

论转基因动物

生物科学发展到二十世纪七十年代时,出现了将外源基因(DNA 片段)插入动物染色体的一门新兴科学——基因转移学,由此而产生了转基因技术和它的产物一转基因动物。1980年首次把外源基因转移给小白鼠的实验获得成功,以后给猪、羊、牛、鱼、兔子等动物转移基因实验也获得了成功,从而转基因动物问世了。转基因动物是生物工程技术的产物,生物工移技术;遗传工程.细胞工程,酶工程和发酵工程所组成,其中的核心是遗传工程,也就是对基因进行剪裁,拼接,改造和加工,按照人们预先设计的蓝图产生特定的性状、品种、物种和产品。     

转基因猪研究的回顾及今后发展方略之我见

简要回顾了20多年来国内外转基因猪的研究,在对猪生产性能的遗传改良、用猪作为生物反应器生产医用蛋白以及用猪作为异种器官移植的供者等方面．取得的一些重要进展：概述了原核注射法、精子介导法、逆转录病毒感染法和体细胞核移植法转基因技术的建立和发展历程：对转基因猪的遗传稳定性进行了讨论。并根据国内外发展的态势和我国养猪业的现状．提出了转基因猪研究方向和技术路线的发展方略．     

国家支持农业转基因技术

日前,科技部部长徐冠华在国务院新闻办举行的记者招待会上说,国家已经在"十五”的863计划中,专门设立生物和现代农业技术领域,来支持植物转基因研究. 　　徐冠华部长进一步介绍了我国农业高新技术的现状:农业生物技术方面,目前已经克隆各种基因近100个,建立了适应多种主要农作物的遗传改良技术体系,应用这些技术,已经获得了具有不同性状的转基因植物180多个,其中进入中试或者大田试验的有15种.我国的抗病毒转基因烟草已经较大面积种植,大约有160万hm2.另外,抗虫棉也在比较大的面积上应用.同时,在抗虫水稻、抗病马铃薯、抗虫玉米方面也都进入了田间试验阶段.在胚胎细胞克隆和连续克隆牛、羊、猪、兔等动物方面都取得了成功.在转基因研究方面,获得了转基因小鼠、猪、牛、羊等动物.另外也获得携带人干扰素基因的转基因兔,携带牛生产激素的转基因猪,培育乳腺高蛋白的转基因牛等. 　　就农业转基因技术,徐冠华进一步解释说,从纯技术的层面来看,转基因技术是中性的,对人体不存在利弊的问题.但是由于转基因食品是把一种外援的基因转移到生物体当中,所以可能有潜在的威胁.这些潜在的威胁往往在短时间内不一定能够发现,因此要加强这方面的监管审批,尽快把这个问题纳入法制化轨道. 　　(本刊辑)     

转基因动物的研究现状与应用前景

通过对转基因动物研究状况、应用及存在问题进行了较为系统的介绍和分析,并结合我国转基因动物研究的现状,对我国发展转基因动物提出了相应的建议,以期促进我国转基因技术的进一步发展,推进我国转基因技术的产业化进程.     

基因编辑猪的研究现状

猪是重要的农业经济动物,猪肉是人类获得蛋白质营养物质的主要途径之一。与此同时,猪在解剖学、生理学及遗传背景、疾病特征等方面与人类极为相似。因此,猪既是农业动物生产性状改良的重要对象,又是人类疾病、异种器官移植等生物医学领域的研究对象。随着基因编辑技术的飞速发展,出现了越来越多操作简单、运用广泛且安全的新型编辑技术,可以快速获得单碱基编辑、基因敲除或敲入的细胞系,并通过体细胞克隆等技术获得基因编辑猪。综述了基因编辑猪的制备及其在农业及医学领域中的研究进展,以期为猪的农业生产性状改良和医学研究提供参考。     

小型猪体细胞克隆试验

[目的]建立体细胞克隆小型猪的技术平台并了解克隆效率。[方法]运用体细胞核移植技术,以小型猪胎儿成纤维细胞作为核移植供体细胞,以体外成熟卵母细胞作为核移植受体细胞进行体细胞克隆猪生产,并利用STR序列对所出生的克隆猪进行个体识别。[结果]将重构胚胎移植到5头代孕母猪体内,其中一头代孕母猪成功产下2头克隆小型猪。经过个体识别试验证实克隆小型猪来自供核细胞,与代孕母猪无直接亲缘关系。[结论]克隆小型猪的成功出生表明所建立的技术平台适于小型猪的克隆。     

Cytogenetic profiles of pigs transgenic for gene construct of human somatoliberin

The effect of integration of the human somatoliberin gene on the cytogenetic profiles of transgenic pigs is studied. A high level of chromosome variability of transgenic animals is established. The highest percentage of chromosome aberrations is found in transgenic animals of the second and third generations.     

Gene engineering in swine for agriculture

Domestic pigs are the second most important source of meat world-wide, and the genetic improvement of economic traits, such as meat production, growth, and disease resistance, is a critical point for efficient production in pigs. Through conventional breeding and selection programs in pigs, which are painstakingly slow processes, some economic traits, such as growth and backfat, have been greatly improved over the past several decades. However, the improvement of many polygenetic traits is still very slow and challenging to be improved by conventional breeding strategies. The development of reproductive knowledge and a variety of techniques, including foreign gene transfer strategies, somatic cell nuclear transfer(SCNT) and particularly, recently developed nuclease-mediated genome editing tools, has provided efficient ways to produce genetically modified(GM) pigs for the dramatic improvement of economic traits. In this review, we briefly discuss the progress of genomic markers used in pig breeding program, trace the history of genetic engineering, mainly focusing on the progress of recently developed genome editing tools, and summarize the GM pigs which have been generated to aim at the agricultural purposes. We also discuss the specific challenges facing application of gene engineering in pig breeding, and future prospects.     

Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

Somatostatin （SS） is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus （PRRSV）. A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.     

二花脸和大约克猪早期生长性状的母体效应与基因效应研究

利用二花脸，大约克及其Ｆ１，Ｆ２和回交世代共８个猪群的最小二乘均数，采用多元回归分析程序，对猪的初生重，２０日龄重的母体效应和基因效应进行了研究。结果表明，大约克猪初生重，２０日龄重及６０日龄严重平均比二花脸猪依次高０．４６１３，１．３６４３和２．０８９０ｋｇ。初生重受母体效应影响最大，而到６０日龄时加性基因效应则占主导地位。     

Characterization of NDM-1-producing carbapenemase in Acinetobacter spp. and E. coli isolates from diseased pigs

In recent years, the mobile metallo-β-lactamase (MBL) genes have been found to correspond to one of the most important resistance characters identified in Gram-negative bacteria, severely affecting clinical chemotherapy and threatening public health. The prevalence of mobile MBL genes and their flanking regions in Gram-negative bacteria from diseased pigs in China was investigated. A total of 334 lung samples from diseased pigs were screened for Gram-negative bacteria classified as non-susceptible to meropenem (MIC≥4 mg·L⁻¹). Six isolates, including three Escherichia coli, two Acinetobacter baumanii and one A. calcoaeticus, exhibited MBL production and carried the bla_NDM-1 gene. S1-PFGE and Southern blot analysis showed that the bla_NDM-1 gene was located on the chromosome of one A. baumanii isolate and on plasmids of various sizes in the other five isolates. MIC testing using broth microdilution revealed that all bla_NDM-1-carrying isolates and some of their transconjugants exhibited resistance to almost all β-lactams tested. Whole genome sequencing revealed that the flanking region of the bla_NDM-1 gene from all porcine isolates had high levels of similarity with the corresponding regions in human isolates. One porcine E. coli isolate carrying bla_NDM-1 was typed as ST48, a common sequence type in human E. coli isolates. These results suggest the possibility of human-to-food animal transfer of bla_NDM-1-producing E. coli, highlighting the need for surveillance of carbapenemase producers among bacteria from food animals. In addition, the prudent use of antimicrobial agents to decrease the opportunities for co-selection of carbapenemase genes in food animals is also urgently needed.     

CD163基因敲除大白猪的抗蓝耳病性能和主要生产性能研究

【目的】运用CRISPR/Cas9基因编辑技术和体细胞核移植技术获得CD163基因全敲除(CD163 gene knockout,CD163-KO)的克隆猪,验证该基因敲除大白猪的抗蓝耳病特性,并研究其与野生型大白猪在生理、生产和繁殖性能等方面的差别,评估CD163-KO大白猪的主要生产性能。【方法】用猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)流行毒株NADC30-like对本研究所获得的11头CD163-KO大白猪和5头年龄、体质量相当的野生型大白猪进行攻毒试验。连续14 d观察猪的临床症状,记录直肠温度变化,检测血清中PRRSV病毒含量和抗体水平。14 d后取肺部组织进行切片,通过PRRSV免疫荧光试验观察肺脏感染情况。采集CD163-KO和野生型大白猪肺泡巨噬细胞进行免疫染色,观察肺泡巨噬细胞表面CD163蛋白的表达情况;采集猪外周血单核细胞进行诱导分化,观察CD163-KO与野生型大白猪诱导分化的巨噬细胞对血红蛋白-结合珠蛋白复合物的摄取情况。最后,统计分析CD163-KO与野生型...