Myostatin基因打靶载体的构建及猪胎儿成纤维细胞Myostatin基因的敲除

目的:用缺口修复等技术构建Myostatin(肌肉生长抑制素,MSTN)基因打靶载体,并对大白猪胎儿成纤维体细胞进行转染,获得基因敲除细胞。方法与结果:首先构建用于MSTN基因同源长臂(LA)的抓捕载体,然后在大肠杆菌内利用Red同源重组系统介导的缺口修复,从含大白猪MSTN基因座的细菌人工染色体上亚克隆9.9 kb的LA到抓捕载体上,经过部分序列测定,同源性为100%;通过PCR获得1.4 kb的同源短臂(SA);将LA和SA连入载体pLOXP,构建含有neo和tk正负筛选标记基因的MSTN基因打靶载体pLOXP-MSTN-KO;将线性化的pLOXP-MSTN-KO通过电转染整合到大白猪胎儿成纤维细胞基因组中,利用G418和丙氧鸟苷进行药物筛选,获得抗性细胞克隆890个,通过PCR和DNA测序鉴定获得基因敲除的细胞克隆4个。结论:构建了有效的MSTN基因打靶载体,通过转染获得基因敲除细胞,为利用体细胞核移植制备MSTN基因敲除猪奠定了基础。     

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。     

CRISPR/Cas9技术制备猪肌肉生长抑制素基因敲除细胞

采用CRISPR/Cas9技术制备质粒HRX-2MCS和PX330,两种质粒转染湖北白猪胎儿成纤维细胞,G418筛选抗性细胞株。收集培养细胞提取总DNA,分子定位PCR法检测绿色荧光蛋白(GFP)基因在猪肌肉生长抑制素(MSTN)基因外显子上的定位整合情况,得到T1靶位点整合细胞5株和T3靶位点整合细胞3株。Q-PCR定量分析结果表明3108号阳性细胞株MSTN基因m RNA表达量减少50%,实验获得的基因敲除细胞可以用于体细胞核移植法制备MSTN基因敲除猪的研究。     

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。     

绵羊MSTN多克隆抗体的制备及细胞毒性分析

目的:研究绵羊肌肉生长抑制素(Myostatin,MSTN)多克隆抗体的安全性。方法:将绵羊MSTN成熟肽基因克隆至原核表达载体pET-32a(+),转化至BL21(DE3)中;利用Ni-NTA亲和层析法纯化目的蛋白,免疫新西兰兔制备多克隆抗体,West-ern blotting鉴定其免疫原性;ELISA检测抗体效价;通过检测乳酸脱氢酶(lactic dehydrogenase,LDH)的水平研究其细胞毒性。结果:成功获得了330 bp的绵羊MSTN成熟肽基因,表达蛋白分子量约为33 kDa,纯度达90%以上;Western blot检测表明MSTN蛋白具有免疫原活性;ELISA检测证实制备的抗体可与抗原蛋白特异性结合,抗体效价为l:25 600,细胞毒性试验表明高浓度的多克隆抗体(200μg/mL)对肌肉细胞无毒害作用。结论:制备的绵羊MSTN多克隆抗体具有生物学活性,对肌肉细胞无细胞毒性,为促生长效果验证提供了安全保障。     

利用启动子缺陷型打靶载体敲除五指山小型猪GGTA1基因

目的:获得α-1,3-半乳糖基转移酶(GGTA1)基因敲除的五指山小型猪胎儿,为异种器官移植研究提供基础平台。方法:以新霉素磷酸转移酶基因(neo)为筛选标记基因构建启动子缺陷型打靶载体,打靶载体线性化后用电转染法将其导入胎儿成纤维细胞中,转染后的细胞经G418筛选后,用PCR法检测药物抗性的细胞克隆,对阳性细胞进行核移植构建重构胚胎及胚胎移植。结果:转染后,经筛选共得到176个具有G418抗性的细胞克隆,经PCR检测,其中2个细胞克隆发生了同源重组;以其中1个GGTA1+/-细胞克隆为供体细胞进行核移植,将重构胚移植到2头自然发情的受体母猪中,1头受体妊娠;第37 d将代孕母猪处死,获得2个胎儿,经PCR和Southern印迹鉴定,均为GGTA1单等位基因敲除胎儿。结论:构建了五指山小型猪GGTA1基因部分外显子4区域敲除的启动子缺陷型打靶载体,获得了GGTA1单等位基因敲除的胎儿,为培育GGTA1基因敲除的五指山小型猪奠定了基础。     

利用CRISPR/Cas9技术建立OXTR基因敲除猪胎儿成纤维细胞系

利用CRISPR/Cas9系统建立催产素受体(oxytocin receptor,OXTR)基因敲除的巴马猪胎儿成纤维细胞系(porcine fetal fibroblasts,PFFs),为构建OXTR基因敲除巴马猪模型提供供体细胞。首先,对人和猪的OXTR基因进行了生物学信息分析。其次,利用在线设计工具设计了2个靶向猪OXTR外显子区的sgRNA,并克隆至pX330骨架质粒中,最后将打靶质粒和Neomycin抗性质粒共转染至PFFs中,用G418药物筛选出抗性单克隆细胞,测序鉴定其基因型。生物信息学分析显示人和猪OXTR基因进化距离较近,氨基酸序列一致性达到91%,相似性为93%,三维结构的RMSD值为0.009。成功构建OXTR基因的Cas9/sgRNA打靶载体,转染PFFs细胞后,药物筛选获得OXTR基因敲除的单克隆细胞并测序证实了基因突变型。人和猪OXTR基因具有高度同源性;构建的Cas9/sgRNA表达载体高效实现了OXTR基因编辑,成功获得基因敲除的单细胞克隆,为后续构建OXTR敲除的巴马猪模型奠定了前期基础。     

过表达组蛋白H3K9me3去甲基化酶对猪克隆胚胎发育的影响

组蛋白异常修饰是克隆胚胎发育的重要制约因素，组蛋白H3K9me3去甲基化酶KDM4家族的过表达可以有效提高克隆胚胎的发育效率。为探究过表达H3K9me3去甲基化酶对猪克隆胚胎发育的影响，本研究在猪克隆胚胎1-细胞期和2-细胞期分别注射KDM4A mRNA和KDM4D mRNA检测胚胎的囊胚率；收集1-细胞期注射KDM4A mRNA和胚胎注射水(对照组)的2-细胞期克隆胚胎检测H3K9me3表达水平；此外，收集1-细胞期注射KDM4A mRNA和胚胎注射水的4-细胞期克隆胚胎进行单细胞转录组测序，并对测序数据进行GO与KEGG富集分析。结果显示：在1-细胞期注射KDM4A mRNA的猪克隆胚胎囊胚率显著高于对照组(25.32±0.74%vs14.78±0.87%)，注射KDM4D mRNA对猪克隆胚胎囊胚率无明显作用(16.27±0.77%vs 14.78±0.87%)；在2-细胞期注射KDM4A mRNA和KDM4D mRNA的克隆胚胎囊胚率与对照组相比均无显著差异(32.18±1.67%、30.04±0.91%vs 31.22±1.40%)。在1-细胞期注射KDM4A mRNA的克隆...     

基于CRISPR/Cas9系统的人成神经瘤SK-N-SH细胞CAPNS1基因的靶向敲除

利用CRISPR/Cas9技术靶向构建CAPNS1基因敲除人成神经瘤细胞SK-N-SH细胞系。在NCBI数据库查找人CAPNS1基因,获得该基因CDS区,根据CRISPR/Cas9敲除靶位点设计原则即g RNA设计原则,设计3条sg RNAs,以p GK1.1为载体,构建人CAPNS1基因敲除载体,并运用共转染的方法转染到SK-N-SH细胞,构建CAPNS1基因敲除SK-N-SH细胞系。结果发现,菌落PCR筛选均能扩增出100 bp大小的片段,单克隆为阳性,DNA测序显示sg RNA序列正确插入质粒,序列比对结果正确;质粒转染SK-N-SH细胞后,制备单克隆,CruiserTMEnzyme酶切后疑似为阳性克隆,进一步的单克隆测序结果显示CAPNS1基因敲除SK-N-SH细胞系构建成功;此外,在CAPNS1-/-组,CAPNS1蛋白及calpain1和calpain2蛋白水平明显降低。以上结果表明CAPNS1基因敲除SK-N-SH细胞系构建成功。     

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.     

Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN-1 and -2

Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple vertebrate MSTNs suggested at least two separate gene duplication events in the fish lineage. Linkage analysis of polymorphic microsatellites within intron 2 of salmon MSTN-1a and -1b mapped the two genes to different linkage groups in agreement with the tetraploid origin of the duplicated salmonid MSTN-1 and MSTN-2 genes.     

Effect of Co-transfection of Anti-myostatin shRNA Constructs in Caprine Fetal Fibroblast Cells

Knockdown of myostatin gene (MSTN), transforming growth factor-β superfamily, and a negative regulator of the skeletal muscle growth, by RNA interference (RNAi), has been reported to increase muscle mass in mammals. The current study was aimed to cotransfect two anti-MSTN short hairpin RNA (shRNA) constructs in caprine fetal fibroblast cells for transient silencing of MSTN gene. In the present investigation, approximately 89% MSTN silencing was achieved in transiently transfected caprine fetal fibroblast cells by cotransfection of two best out of four anti-MSTN shRNA constructs. Simultaneously, we also monitored the induction of IFN responsive genes (IFN), pro-apoptotic gene (caspase3) and anti-apoptotic gene (MCL-1) due to cotransfection of different anti-MSTN shRNA constructs. We observed induction of 0.66-19.12, 1.04-4.14, 0.50-3.43, and 0.42-1.98 for folds IFN-β, OAS1, caspase3, and MCL-1 genes, respectively (p < 0.05). This RNAi based cotransfection method could provide an alternative strategy of gene knockout and develop stable caprine fetal fibroblast cells. Furthermore, these stable cells can be used as a cell donor for the development of transgenic cloned embryos by somatic cell nuclear transfer (SCNT) technique.     

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.     

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector‐mediated RNAi

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin‐signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector‐based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic‐cell nuclear transfer (SCNT) studies. Sh‐RNA positive cells were screened by puromycin selection. Using real‐time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down‐regulation in sh2 shRNA‐treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin‐targeting siRNA produced endogenously could efficiently down‐regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus‐mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:452–459, 2015     

Nuclear reprogramming in embryos generated by the transfer of yak (Bos grunniens) nuclei into bovine oocytes and comparison with bovine-bovine SCNT and bovine IVF embryos

Although inter-species SCNT may be useful for increasing and preserving populations of endangered species, there are many reports that inter-species nuclear transfer embryos only develop to the blastocyst stage. In this study, yak-bovine SCNT blastocysts were successfully implanted in the surrogate bovine uterus but failed to develop to term or aborted. To clarify the reasons, we examined yak-bovine SCNT blastocyst development, total cell number, inner cell mass (ICM) number, trophoblast (TE) cell number and relative gene expression in yak fibroblast cells and yak-bovine SCNT embryos at various stages. The potential for development of yak-bovine SCNT embryos to blastocysts was 30+/-5.7% (mean+/-S.E.M.); the total cell number was 85.3+/-16.3, fewer than in IVF bovine embryos (106.2+/-18.2) but within the reported range (60-300). The yak-bovine SCNT blastocysts had a lower ratio of TE cells to total cells (43.9+/-8.7%) than bovine IVF embryos (59.4+/-3.4%; P<0.05) or bovine-bovine SCNT (69.5+/-5.4%; P<0.05). Also, several yak-bovine SCNT embryos had abnormal initiation of expression of both Mash2 and IL6. However, expression of vimentin, collagen, Cx43 and PSMC3 were normal in yak fibroblast cells and yak-bovine SCNT embryos. In conclusion, we inferred that the normal allocation of ICM and TE cells in yak-bovine SCNT embryos and embryo-specific gene reprogramming may be important for successful inter-species animal cloning.