利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因

肌肉生长抑制素(Myostatin, MSTN)基因能够负向调节骨骼肌的生长和发育, 牛MSTN基因突变会出现“双肌”特征。文章利用转录激活因子样效应物核酸酶(TALENs)靶向敲除牛胎儿成纤维细胞的MSTN基因, 获得敲除MSTN基因的细胞系, 为制备MSTN基因敲除牛提供材料。构建一对MSTN基因的TALENs真核表达载体, 分别采用PEI转染试剂和电穿孔法进行牛胎儿成纤维细胞的转染, 测序结果表明TALEN技术可用于敲除牛MSTN基因, 利用T7核酸内切酶1(T7E1)检测其突变效率, 结果显示电穿孔转染的敲除效率为20.4%。通过有限稀释法, 共获得10个MSTN基因敲除的细胞克隆(包括MSTN^-/-和MSTN^+/-), 其靶位点敲除的碱基数分别是1~20不等, 部分会出现移码突变。出现移码突变的细胞系可用于MSTN基因敲除的转基因肉牛的制备。     

TALENs介导MSTN基因突变山羊的制备及性能分析

肌肉生长抑制素(myostatin, MSTN)是一种骨骼肌生长发育的负调控因子,可以抑制成肌细胞的增殖,是动物品种改良的重要候选基因,该基因突变能够引起骨骼肌的广泛性增生与肥大,产生“双肌”症状,导致动物脂肪分化减少、肌肉含量增加,从而满足市场动物肉品质消费的需求。为了获得“双肌”表型的MSTN基因突变克隆山羊,本研究在山羊MSTN基因第一外显子序列设计并构建TALENs表达载体,转染山羊胎儿成纤维细胞,经嘌呤霉素筛选获得抗性细胞株,通过PCR检测和基因测序筛选MSTN基因突变细胞株作为供核细胞进行山羊体细胞核移植,并鉴定MSTN基因突变类型;通过组织切片分析MSTN基因突变山羊肌肉组织形态结构,监测不同月龄克隆山羊体重并分析其不同生长发育阶段的体重增长趋势。结果表明,共获得109株MSTN基因突变细胞株,突变效率达到79.0%(109/138),其中46株为双等位基因突变,占细胞株总数的33.3%(46/138)。选取4株生长状态较好的MSTN基因突变细胞株(1株为双等位基因纯合突变,3株为非纯合突变)进行体细胞核移植至12只受体山羊,30d时B超检查出受孕母羊4只,受孕率为33....     

MSTN基因突变纯合子兔的繁育和表型分析北大核心CSCD

肌肉生长抑制素(myostatin,MSTN)基因是动物骨骼肌生长的负调控基因。本研究在获得MSTN基因双等位突变原代兔的基础上,培育具有“双肌”表型性状和稳定遗传的MSTN^(–/–)纯合突变兔,建立原代MSTN双等位突变兔繁育纯合子后代的方法。应用CRISPR/Cas9基因编辑技术制备MSTN–/–突变原代兔,原代双等位突变兔与野生型兔配种,产生F1代兔。同一突变系的F1代兔半同胞交配或回交繁殖F2代纯合子兔;后代兔利用基因组DNA PCR扩增产物序列分析和T载体克隆测序鉴定用于筛选纯合子兔;14–19周龄MSTN基因突变兔称量体重。MSTN基因突变兔臀大肌组织切片,统计分析肌纤维横截面积。获得5只MSTN基因突变原代兔,其中3只用于纯合子培育,共获得15只(5种突变子)纯合突变兔(M2-a:3只;M2-b:2只;M3-a:2只;M7-a:6只;M7-b:2只)。在14–19周龄,MSTN^(–/–)纯合突变兔体重极显著高于同龄MSTN+/+野生型兔(平均(2718±120)g和(1969±53)g,P<0.01,同龄体重提高38.0%);纯合突变兔的臀大肌纤维平均截面积极显著高于野生型兔((3512.2±439.2)μm^(2)和(1274.8±327.3)μm^(2),P<0.01),也显著高于MSTN^(+/–)半合子兔((3512.2±439.2)μm^(2)和(2610.4±604.4)μm^(2),P<0.05)。本研究成功培育了5种MSTN^(–/–)基因纯合突变兔,且有明显瘦肉表型。研究表明,应用CRISPR/Cas9基因编辑获得的原代双等位突变兔为非嵌合体突变兔,突变性状遗传后代;原代双等位突变兔后代分离为两个不同突变子,并都呈现“双肌”表型;F1代单等位突变兔通过近交或回交可获得MSTN^(–/–)纯合突变兔。该研究在利用基因编辑技术培育优良家畜品种方面取得了实验进展。     

猪肌肉生长抑制素基因5′调控区T→A突变与生长性状的关系分析

生长速度是猪育种中选择的主要目标性状之一,鉴定影响生长速度的QTL或基因具有重要的实践意义和理论价值.对猪肌肉生长抑制素基因(myostatin,MSTN)5′调控区TA突变与生长性状的关系进行了分析.突变所产生的基因型的判定采用PCR-RFLP方法进行.猪生长性状的测定指标包括60日龄体重(body weight at 60d,BW60)、前期平均日增重(average daily gain of stage one,ADG1)、后期平均日增重(average daily gain of stage two,ADG2)和全期平均日增重(average daily gain of whole stage,ADG),所采用的记录数分别来自165,276,275和275头无亲缘关系个体.采用SAS软件包的GLM程序对MSTN基因型与猪生长性状的关系进行分析.结果表明,MSTN基因型对猪BW60,ADG1和ADG2的效应不显著(P＞0.1),对ADG2的效应在剔除基因型与品种的互作效应以后几乎达到显著水平(P＝0.0522),突变等位基因的携带者(基因型为TA)具有较高的后期平均日增重.     

基因打靶定点突变秦川牛MSTN基因

Myostatin(MSTN,肌肉生长抑制素)基因属于TGF-β超家族,对骨骼肌的生长发育具有负调控作用。该基因的功能缺失,能够引起肉用动物的"双肌"表型,从而提高产肉率。基因打靶技术是制作转基因动物的常用方法。构建了两个置换型打靶载体pA2T-Mstn4.0和pA2T-Mstn3.2,通过同源重组将G938A突变点引入秦川牛MSTN基因第三外显子。电穿孔方法转染秦川牛胎儿成纤维细胞,经过600μg/mL G418和50nmol/L GCV的药物正负筛选,共得到170个药物抗性细胞克隆。对细胞克隆进行PCR、测序及Southern blotting鉴定,结果显示,第58号细胞克隆为发生了正确同源重组的中靶细胞。牛胎儿成纤维细胞中的MSTN基因的一条等位基因被成功改造。     

肌肉生长抑制素调控动物骨骼肌生长机制的研究进展

肌肉生成抑制素（myostatin, MSTN）在动物机体骨骼肌的增殖、分化和生长中起着重要的负调控作用。MSTN基因的过表达会阻碍骨骼肌增殖分化及生长发育,而缺失或表达降低则会导致肌肉肥大,形成双肌现象（double muscle phenomenon, DMP）。MSTN能作用于多个基因及结合多种细胞因子广泛参与生理生化、物质代谢、病理调控等过程,在动物机体生长发育过程中扮演着重要的角色。本文将从MSTN基因的历史渊源、基因定位、时空表达特性、部分相关作用机制等方面进行论述,旨在对MSTN调控动物骨骼肌生长部分机制作梳理,以期为后期研究提供理论依据。     

甘肃西门塔尔牛甘州类群MSTN基因外显子多态性分析

采用PCR-SSCP方法检测50头甘肃西门塔尔牛甘州类群MSTN基因3个外显子的遗传多态性,并对群体内各等位基因进行测序,旨为探讨西门塔尔牛MSTN基因与肉质性状关联等方面的研究提供理论依据。结果显示,甘肃西门塔尔牛甘州类群MSTN基因3个外显子中,外显子2受B、C等位基因控制,形成BB、CC和BC 3种基因型,基因型频率分别为0.22(11/50)、0.64(32/50)、0.14(7/50);外显子1和外显子3分别受A等位基因与D等位基因控制,形成AA和DD基因型,基因型频率均为1(50/50)。序列分析表明,甘肃西门塔尔牛甘州类群MSTN基因3个外显子中,外显子2在第41 bp处发生了C→T的突变,但并没有导致氨基酸发生改变,属于同义突变;外显子1和3均无突变。统计结果表明,甘肃西门塔尔牛甘州类群MSTN基因3个外显子中,外显子2为中度多态,其余均无变化。     

黄河裸裂尻鱼肌肉生长抑制素基因克隆及表达分析

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子β(TGF-β)超家族中的一个成员,是骨骼肌生长发育的负调控因子。该文以黄河裸裂尻鱼肌肉总RNA为模板,采用RT-PCT、5’-RACE和3’-RACE法获得MSTN基因全长cDNA序列为2180bp,包含长为1128bp的开放阅读框,编码375个氨基酸。以肌肉总DNA为模板,通过PCR法进一步获得了MSTN基因的2个内含子序列,分析表明,黄河裸裂尻鱼MSTN基因与其他脊椎动物具有相似的基因结构(包括3个外显子和2个内含子)。黄河裸裂尻鱼MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RXXR和8个位于TGF-β功能区域保守的半胱氨酸残基。氨基酸序列同源性分析表明,黄河裸裂尻鱼MSTN序列与其他鲤科鱼类MSTN具有较高的同源性;而与哺乳动物和禽类的MSTN同源性较低。系统发育分析表明,黄河裸裂尻鱼MSTN与其他鲤科鱼类聚于同一进化支。RT-PCR分析表明,该基因在黄河裸裂尻鱼9个被检组织中均有表达,但在心、肾、肠、精巢中表达量较高。Real-TimePCR分析显示,MSTN基因在胚胎中的相对表达量,随胚胎发育阶段的不同而有所差异,暗示MSTN的功能可能并不局限在对肌肉生长发育的负调控作用,可能还有其他功能。     

肌肉生长抑制素基因在哺乳动物中的最新研究进展

肌肉生长抑制素（myostatin,MSTN）,也称为生长分化因子8（GDF8）,是转化生长因子β（TGF-β）超家族成员,主要在骨骼肌中表达,在负向调控肌细胞的生长发育中起关键作用。本文对MSTN研究的最新进展进行了综述,讨论了MSTN信号通路与其他通路之间的相互关系,重点阐述了试验条件下MSTN对肌肉发育相关基因的作用,介绍了通过基因操作或使用抗体的方法抑制MSTN的表达对于提高动物产肉量、治疗肌肉萎缩等疾病以及延缓衰老的重要意义。     

myostatin基因打靶的成肌细胞制备

Myostatin(MSTN), β-转化生长因子超家族的一员,是肌肉生长的负调控因子,该基因自然突变的比利时蓝牛和皮尔蒙特牛出现双肌性状。基因敲除该基因,是实现家畜产肉量的提高的有效方法。通过建立无启动子打靶载体MSTN-GFP, MSTN-neo,转染新生和胎儿绵羊骨骼肌细胞,经过表达绿色荧光蛋白报告基因和G418筛选获得骨骼肌细胞克隆,PCR,Southern blot,DNA序列检测获得阳性细胞克隆,为制备myostatin基因敲除的体细胞克隆绵羊提供核移植供体。     

Mutations in the Myostatin gene leading to hypermuscularity in mammals: indications for a similar mechanism in fish?

The transforming growth factor β (TGF-β) superfamily encodes secreted factors that are important in regulating embryonic development and tissue homeostatis in adults. Myostatin (MSTN, encoded by MSTN) or 'growth and differentiation factor 8', a member of this superfamily, is a negative regulator of skeletal muscle growth and is highly conserved among animal species. In 1997, a mutation associated with the so-called double-muscling phenotype in cattle was found in the MSTN gene. During the years following the discovery of the first MSTN mutation, other mutations were found in cattle and other mammalian species, and MSTN became one of the most thoroughly studied genes in animals. The aim of this review is mainly to describe the functional mutations located in the MSTN genes of several mammalian species, leading to double muscling in these animals. Furthermore, in light of the increasing importance of fish genetics, the possibility of functional mutations in piscine MSTN with a similar effect as in mammals, and a genetic model for MSTN research in fish, will also be discussed.     

Associations between the 11-bp deletion in the myostatin gene and carcass quality in Angus-sired cattle

An 11-bp deletion in the bovine myostatin ( MSTN ) gene was identified as the causative mutation for the double-muscling phenotype in Belgian Blue and Asturiana cattle. More recently, this mutation was also identified in the South Devon breed of cattle, in which it has been found to be associated with a general increase in muscle mass. The present study found that the mutant allele was also segregating in a commercial population of Scottish Aberdeen Angus beef cattle. The mutation was found at a low frequency (0.04) with no animals homozygous for the mutation in the sample population (536 animals). The effects of this mutation on various carcass traits of economic interest were then tested. We found that the mutation significantly increased carcass weight, sirloin weight, hindquarter weight, muscle conformation score and eye muscle area, but had no effect on the fat traits.     

Effect of myostatin F94L on carcass yield in cattle

In this study, a highly significant quantitative trait locus (QTL) for meat percentage, eye muscle area (EMA) and silverside percentage was found on cattle chromosome 2 at 0-15 cM, a region containing the positional candidate gene growth differentiation factor 8 (GDF8), which has the common alias myostatin (MSTN). Loss-of-function mutations in the MSTN gene are known to cause an extreme 'double muscling' phenotype in cattle. In this study, highly significant associations of MSTN with cattle carcass traits were found using maternally inherited MSTN haplotypes from outbred Limousin and Jersey cattle in a linkage disequilibrium analysis. A previously reported transversion in MSTN (AF320998.1:g.433C>A), resulting in the amino acid substitution of phenylalanine by leucine at position 94 of the protein sequence (F94L), was the only polymorphism consistently related to increased muscling. Overall, the size of the g.433C>A additive effect on carcass traits was moderately large, with the g.433A allele found to be associated with a 5.5% increase in silverside percentage and EMA and a 2.3% increase in total meat percentage relative to the g.433C allele. The phenotypic effects of the g.433A allele were partially recessive. This study provides strong evidence that a MSTN genotype can produce an intermediate, non-double muscling phenotype, which should be of significant value for beef cattle producers.     

Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity

Myostatin (MSTN), a transforming growth factor beta superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5' and 3' UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks.     

中国肉牛分子与基因修饰育种研究进展

随着世界肉牛产业科技的快速发展,我国肉牛产业的整体水平得到明显提高并取得丰硕成果。肉牛育种技术实现了由常规杂交育种向分子标记辅助育种、全基因组选择育种和基因组修饰育种的技术跨越,揭示出大量与生长发育、肉质品质、繁殖与疾病等相关的候选基因与分子标记,并逐步应用于肉牛育种实践。与生长发育性状相关的基因或分子标记主要集中在生长激素基因、生肌调节因子家族、肌肉生长抑制因子和胰岛素样生长因子等;参与肉质形成的基因主要集中在脂肪酸运输与沉积相关信号通路、钙蛋白酶信号通路、生肌调节因子家族与肌肉生长抑制因子等;繁殖性状相关基因或分子标记主要集中在GnRH-FSHR-LH、生长分化因子9、催乳素受体和FoxO1等;抗病相关基因主要有MHC基因家族、TOLL样受体4基因等。目前,利用精准基因编辑技术已培育出促生长发育与提高肉品质的肉牛育种新材料。本文总结了近年来我国在肉牛分子与基因组修饰育种领域取得的研究进展,以期为我国肉牛遗传育种技术研究提供参考和借鉴。     

军曹鱼肌生成抑制素基因的克隆与序列分析

肌生成抑制素(Myostation,MSTN)是一种骨骼肌生长的负调控因子,其生物功能主要是抑制骨骼肌的生长。肌生成抑制素的活性降低或丧失,可使肌肉与其他组织的比例大大提高,因此在动物育种和医疗上有很大的潜在应用价值。目前包括鱼类在内的20多种脊椎动物的MSTN cDNA已经得到克隆和测序。本实验依据已知的鱼MSTN cDNA的保守区域设计一对特异引物,利用PCR技术分别从军曹鱼基因组中扩增出一个约1000bp的特异片段和300bp片段,所得目的片段回收纯化,将其酶切产物连接到pMDl8-T克隆载体上,转化入JM109感受态细胞中,挑取阳性克隆进行转化子鉴定,其质粒测序结果与文献报道的一致,证明成功地克隆了军曹鱼肌生成抑制素基因。     

Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.     

Association of the expression levels in the skeletal muscle and a SNP in the CDC10 gene with growth‐related traits in Japanese Black beef cattle

Growth performance, as well as marbling, is the main breeding objective in Japanese Black (JB) cattle, the major beef breed in Japan. The septin 7 (CDC10) gene, involved in cellular proliferation, is located within a genomic region of a quantitative trait locus for growth‐related traits. In this study, we first showed that the expression levels of the CDC10 gene in the skeletal muscle were higher in JB steers with extremely high growth performance than in JB steers with extremely low growth, using real‐time PCR. Further, a single nucleotide polymorphism (SNP), NC_007302.5:g.63264949G>C, was detected in the promoter region of the CDC10 gene and genotyped in three Japanese cattle breeds (known as ‘Wagyu’ in Japan) and the Brown Swiss dairy cattle breed. All four cattle populations showed a moderate genetic diversity at the SNP of the CDC10 gene. An association analysis indicated that the SNP was associated with growth‐related traits in JB cattle. These findings suggest possible effects of the expression levels in the skeletal muscle and the SNP of the CDC10 gene on growth‐related traits in JB cattle. The CDC10 SNP may be useful for effective marker‐assisted selection to increase beef productivity in JB beef cattle.