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     

无标记转Fat-1基因真核表达载体的构建及转基因绵羊细胞系的建立

为了建立无筛选标记基因的转Fat-1基因绵羊细胞系,本研究将PCR克隆得到的Fat-1基因,合成的attB、Loxp序列并克隆入pN1-EGFP框架载体,得到可删除筛选标记基因的pEGFP-N1-Fat-1真核表达载体。体外转录合成phiC31整合酶mRNA并与线性化的pEGFP-N1-Fat-1载体共转染绵羊胎儿皮肤成纤维细胞,G418筛选得到表达绿色荧光的单克隆,再利用pET-28a-His-NLS-TAT-Cre质粒诱导Cre重组蛋白表达,将纯化后的Cre穿膜肽转导表达绿色荧光的单克隆细胞,将荧光淬灭的细胞系扩繁,提取基因组DNA,进行PCR及测序鉴定,得到无标记转Fat-1基因绵羊胎儿皮肤成纤维细胞系,为生产无筛选标记基因的转基因绵羊奠定基础。     

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells

Myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-β) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.     

Knockdown of Myostatin Expression by RNAi Enhances Muscle Growth in Transgenic Sheep

Myostatin (MSTN) has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT) technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production.     

Production of transgenic calves expressing an shRNA targeting myostatin

Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.     

卵周隙内注射慢病毒生产转基因绵羊的方法

目的提高并稳定转基因绵羊生产效率。方法采用卵周隙内注射慢病毒的方法生产转基因绵羊。比较了不同发育阶段卵母细胞注射慢病毒对其后期发育及基因表达的影响,对照了注射不同慢病毒剂量以及不同熟练程度人员操作,对胚胎发育及基因表达率的影响。结果 (1)受精前或受精后注射慢病毒对胚胎后期发育和阳性率没有影响(P>0.05);(2)在受精后的单细胞注射或者2～4细胞期注射慢病毒对胚胎后期发育和阳性率也都没有影响(P>0.05);(3)慢病毒注射剂量在50～100 pL之间对转基因效率没有影响(P>0.05);(4)操作人员的熟练程度不影响胚胎成活率和转基因效率(P>0.05)。结论建立了卵周隙内注射慢病毒生产转基因绵羊的方法,并使转基因胚胎阳性率稳定在70%以上。     

Knockdown of endogenous myostatin promotes sheep myoblast proliferation

Myostatin (MSTN), is a known negative regulator of myogenesis. Silencing of the function of MSTN could result in increasing muscle mass in mice. To determine the function of endogenous MSTN expression on proliferation of sheep myoblasts, a short-hairpin RNA-targeting sheep MSTN was constructed into lentiviral vector to silence endogenous MSTN expression. We demonstrated that silencing of endogenous MSTN gene with up to approximately 73.3% reduction by short hairpin RNA (shRNA) resulted in significant increase (overall 28.3%) of proliferation of primary ovine myoblasts. The upregulation of proliferation was accompanied by the decrease expression of MyoD (?37.6%, p?=?0.025), myogenin (?33.1%, p?=?0.049), p21 (?49.3%, p?=?0.046), and Smad3 (?50.0%, p?=?0.007). Silencing of myostatin using shRNA may provide a feasible approach to improve meat productivity in farm animals.     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (p?=?0.003) and 54% (p?=?0.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, p?=?0.001), MYOG (94%, p?=?0.000), and MYF5 (36%, p?=?0.000), cell cycle regulator p21 (62%, p?=?0.000), MSTN receptor ACVR2B (23%, p?=?0.061), MSTN antagonist follistatin (81%, p?=?0.000), and downstream signaling mediators SMAD2 (20%, p?=?0.060) and SMAD3 (49%, p?=?0.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (p?=?0.354) and 79% compared to untransduced myoblasts (p?=?0.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; p?=?0.000)- and sh2 (1.71-fold; p?=?0.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.     

CRISPR/Cas9介导的同源重组技术构建猪肌抑素基因敲除细胞系

肌抑素是肌肉增生的负调控因子,为改良家畜产肉性状的重要候选基因。本研究设计了Cas9/sgRNA表达载体与供体DNA,通过电转染方法将其导入猪PK15细胞,经G418抗性筛选和荧光蛋白标记甄别,筛选到带阳性标记的细胞克隆。通过跨界PCR、长距离PCR、Western印迹、Southern印迹及PCR产物测序,证明了猪肌抑素的第3外显子序列特异性同源重组事件的发生。在猪肌抑素的第3外显子区域找到了1个有效的CRISPR/Cas9打靶位点,带阳性标记的细胞克隆经过多次筛选分离,获得了肌抑素单等位基因失活的稳定细胞系,为深入研究肌抑素的功能提供了重要的实验材料。     

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Background

Low efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed.

Methodology/Principle Findings

EGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep.

Conclusions/Significance

Injection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification.     

基于人免疫缺陷病毒-1的慢病毒载体系统研究进展

慢病毒能够感染分裂细胞和非分裂细胞,因而被发展成为重要的转基因载体。慢病毒载体已经发展到了第三代,其安全性已经大为提高。经过结构优化的慢病毒载体已经用于转基因动物生产和基因治疗研究,而稳定包装细胞系的建立使得慢病毒的生产更为简便。     

Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.     

The critical role of myostatin in differentiation of sheep myoblasts

Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.     

用绿色荧光蛋白（GFP）作为报告分子筛选有效的siRNA

 建立一种利用绿色荧光蛋白（GFP）作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子（MIF）基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIF siRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIF siRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.     

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.     

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。     

siRNA干扰HSV1 gD糖蛋白基因的研究

以HSV1 gD糖蛋白基因为靶点,设计合成5对siRNA,并构建pEGFP-N1-gD融合表达质粒,脂质体法共转染Vero细胞,利用绿色荧光蛋白(EGFP)报告基因的特征,流式细胞仪筛选特异沉默gD表达的siRNA。然后有效siRNA转染Vero细胞并感染HSV1,通过空斑减数实验,Real-time PCR和子代病毒滴度评价其对HSV-1感染的作用。结果显示siRNA能有效抑制gD-EGFP融合蛋白和感染细胞内gD糖蛋白的表达,但对HSV-1的感染性无明显抑制作用,故选择gD作为siRNA抗病毒靶点还需进一步的论证。     

In Planta visual monitoring of green fluorescent protein in transgenic rice plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used reporter that can be directly visualized in the living cells in both animals and plants. We inserted a synthetic gene (sgfp) encoding a modified form of the GFP into expression vector, Act1-sgfp for the direct expression of GFP which is easily detectable in rice plants. Green fluorescence emitted from GFP could be visualized in calli, dry seeds, roots and seedlings with green shoots of transgenic rice plants. In our visualization system with a charge-coupled device camera, band-pass filters and a light source, the presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of GFP. These results demonstrate that GFP could be used as a non-destructive visual selection marker for examining gene expression in transformed calli, dry seeds and young plants.     

In vivo gene transfer to mouse spermatogenic cells using green fluorescent protein as a marker

Combination of the DNA injection into seminiferous tubules and the subsequent in vivo electroporation (EP) has become an efficient and convenient assay system for spermatogenic-specific gene expression during spermatogenesis of mice. In this study, we made methodological modifications to enhance the transfection efficiency, and evaluated the possibility of this technique to generate transgenic offspring using green fluorescent protein (GFP) as a marker. After the in vivo gene transfer, GFP expression could be monitored easily and repeatedly on the surface of the testis of live mice under fluorescent microscopy. The serial sections of the transfected testis revealed that transient expression of GFP was extended even in the innermost region of the testis uniformly, but confined to spermatogenic cells and Sertoli cells within the seminiferous tubules. Furthermore, long-lasting GFP expression could be detected in the spermatogenic cells even 2 months after EP. Natural mating with normal adult females revealed that 65% of the transfected males maintained fertilizable ability and could generate their offspring normally. Germ-line transmission of the GFP vector to the offspring was checked under fluorescent microscopy, but no transgenic offspring has been detected up to now. These results suggest that the application of additional techniques, such as cell sorting for GFP-positive germ cells followed by nuclear transfer to the oocytes, would make this method as a novel strategy for generating transgenic animals. J. Exp. Zool. 286:212-218, 2000.