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Engineered zinc finger nucleases (ZFN) are rapidly gaining popularity as a means to enhance the rate and specificity of DNA modifications in plant and animal cells. Repair-mediated gene modification by ZFN is driven by introducing DNA double-strand breaks via a nonspecific nuclease domain linked to a sequence-specific zinc finger nucleotide recognition domain. This review examines the use of ZFN to produce genetically modified swine and the potential of this technology for the future. By combining conventional gene targeting methods with somatic cell nuclear transfer, several genetically modified pig models have been produced. These conventional techniques are inefficient in mammalian somatic cells and provide little control over the site specificity and rate of exogenous DNA integration. The use of engineered ZFN that bind and cleave genomic DNA at specific loci can enhance targeting efficiencies by orders of magnitude. Recent publication of the first genetic modification in pigs by combining ZFN technology with somatic cell nuclear transfer has opened the door to genome targeting with a precision that was not previously possible in a large animal model. Since that time, model pigs with selective knockout of endogenous genes have been produced. This review will examine the use of ZFN to generate these pig models and the potential of ZFN to accelerate the production of genetically modified pigs of agricultural and biomedical importance. Current methods of ZFN design, important considerations for their safe and effective use in modification of the swine genome, and future innovative applications of this technology in pigs will be discussed. 相似文献
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Xiang-Xing Zhu Qun-Mei Zhan Yan-Yan Wei Ai-Fen Yan Juan Feng Lian Liu Sheng-Sheng Lu Dong-Sheng Tang 《Reproduction in domestic animals》2020,55(10):1314-1327
CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed. 相似文献
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肌肉生长抑制素(myostatin,MSTN)基因突变可引起动物出现"双肌"性状,提高产肉性能。利用CRISPR/Cas9技术制备MSTN基因敲除的绵羊胎儿成纤维细胞,为制备MSTN基因敲除羊提供材料。设计构建4个靶向MSTN基因的CRISPR/Cas9载体,脂质体转染细胞后,通过SURVEYOR分析和测序等方法对敲除效率进行检测,采用极限稀释法挑选稳定敲除的细胞系。试验成功构建了4个靶向MSTN基因的CRISPR/Cas9载体,细胞转染后,测序结果显示pX330-target 1和pX330-target 4载体作用的靶位点处出现突变,SURVEYOR分析检测其在靶位点产生切割的效率分别为24.20%和10.18%。通过极限稀释法,获得12个MSTN基因突变的细胞克隆,其中1个为纯合突变。序列比对发现靶位点处有小片段碱基插入或缺失突变,部分会出现移码突变。成功利用CRISPR/Cas9系统实现了绵羊MSTN基因敲除,证明该系统可有效应用于绵羊基因编辑,产生的突变细胞系为制备MSTN基因敲除羊提供了材料。 相似文献
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Mutation in myostatin (MSTN) gene resulted in double muscle effect,generating more mutton.To knock out MSTN gene in sheep fetal fibroblast by CRISPR/Cas9 system and obtain MSTN gene knockout cell lines,four plasmids were designed and constructed to target MSTN gene,and confirmed correctly by sequencing.The correct plasmids were delivered into the fetal fibroblast cells.The targeting efficiency was detected using SURVEYOR assay Kit.The stable transfected cell colonies were obtained via limiting dilution procedure.The sequence results demonstrated that the pX330-target 1 and pX330-target 4 plasmids could successfully knockout MSTN gene,and the targeting efficiency were 24.20% and 10.18%,respectively.Twelve MSTN gene knockout cell colonies were obtained via limiting dilution,and one of them was homozygous mutation.Several indel mutations were discovered at specific site,and some of them were frame-shift mutation.Therefore,we concluded that the CRISPR/Cas9 system could apply to the gene editing of sheep efficiently,and the gene knockout cell clones had potential application in generating MSTN gene knockout sheep. 相似文献
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旨在构建并包装打靶绵羊MSTN基因的位点特异性锌指核酸酶腺病毒表达载体,以借助腺病毒的高转染效率和非整合性以及锌指核酸酶的高效性和特异性实现对绵羊MSTN基因的敲除。本研究利用PCR扩增T2A序列和锌指核酸酶异源二聚体序列,测序鉴定后依次克隆至pAdTrack-CMV,得到pAdTrack-ZFNL-T2A-ZFNR穿梭载体,将之与腺病毒骨架载体pAdEasy-1共转染至BJ5183菌株进行同源重组,以构建pAdEasy-ZFNL-T2A-ZFNR表达载体。然后用上述表达载体转染HEK293细胞进行重组腺病毒的包装,将PCR鉴定阳性的病毒进行扩增并测定病毒滴度。侵染绵羊胎儿成纤维细胞检测重组腺病毒对靶细胞的侵染能力,并用Western blot方法检测绵羊胎儿成纤维细胞中ZFN的表达,进而在细胞水平验证ZFN的活性。结果,包装得到的锌指核酸酶重组腺病毒能够高效侵染绵羊胎儿成纤维细胞并表达ZFN,腺病毒介导的ZFN可识别并切割绵羊MSTN基因。本研究成功获得靶向识别并切割绵羊MSTN基因的锌指核酸酶重组腺病毒。 相似文献
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Generation of α‐1,3‐Galactosyltransferase‐Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9‐Mediated Knock‐in of a Small Mutated Sequence and a Targeted Toxin‐Based Selection System 
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下载免费PDF全文 M Sato A Kagoshima I Saitoh E Inada K Miyoshi M Ohtsuka S Nakamura T Sakurai S Watanabe 《Reproduction in domestic animals》2015,50(5):872-880
The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α‐gal epitope synthesized by α‐1,3‐galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig‐to‐human xenotransplantation. We here employed the CRISPR/Cas9 system‐mediated knock‐in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800‐bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α‐gal epitope expression, we successfully obtained several knock‐in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9‐mediated HR to knock‐in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets. 相似文献
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Jianwen Chen Kaiyuan Pan Zhen Chen Yunsheng Li Biao Ding Chunyang Han Zubing Cao Wenbin Bao Yunhai Zhang 《Animal Science Journal》2019,90(3):366-371
Porcine viral diarrhea is an acute and highly contagious enteric disease in pigs which causes huge economic losses in pig industry worldwide. Transmissible gastroenteritis virus (TGEV) is main pathogens responsible for piglets viral diarrhea. Knockout the host cellular surface receptor for TGEV may be an effective way to accelerate the breeding of resistant pigs. In this study, we applied site‐specific editing pAPN which is effective in swine testis (ST) cells. Site‐specific editing of pAPN reduced TGEV proliferation in ST cells by 96%–99% at different time periods post‐infection. Next, the site‐specific editing of pAPN porcine fetal fibroblasts were produced, and then the cell colonies were used as donor cells to generate the site‐specific editing of pAPN pigs. Our research findings will not only offer a more thorough understanding of the pathogenesis of piglet diarrhea and lay the foundation for breeding TGEV‐resistant piglets, but also understanding the molecular mechanisms involved in coronaviral infections. 相似文献
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基因编辑是利用核酸酶在基因组的特定位点产生DNA双链断裂，从而触发细胞自身的DNA损伤修复机制，实现对靶基因序列进行位点特异性修饰。规律成簇间隔短回文重复序列（clustered regularly interspersed short palindromic repeat，CRISPR）及其相关蛋白9（Cas9）系统作为第3代基因编辑技术在农业和基因治疗研究中发挥着重要作用，与传统的锌指核酶和转录激活因子样效应物核酶基因编辑方法相比，它可以靶向基因的任何位点引起DNA双链断裂，实现目标基因的精准敲除或插入外源片段，并能快速、高效地修饰基因组，包括基因敲除、敲入、抑制、激活等，是应用最广泛的基因编辑工具。CRISPR/Cas9技术的出现彻底改变了生命科学、医学和遗传学研究现状。近年来，利用CRISPR/Cas9技术对动物基因组进行修饰，开启了畜禽分子育种的新纪元，不仅极大地推动了现代畜禽养殖技术的发展，而且为人类医学研究做出了特殊贡献，特别是在猪和鸡上。作者以猪和鸡为对象，综述了CRISPR/Cas9技术在异体器官移植供体、人类疾病的生物模型、抗病育种材料、全基因组高通量筛选等方面的研究进展，以及如何利用CRISPR技术快速又简单地生产出基因编辑猪、鸡，为推动CRISPR技术制备其他基因编辑动物提供参考依据。 相似文献
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J‐T Kang J Ryu B Cho E‐J Lee Y‐J Yun S Ahn J Lee D‐Y Ji K Lee K‐W Park 《Reproduction in domestic animals》2016,51(6):970-978
Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies. 相似文献
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Fuminori Tanihara Maki Hirata Nhien T. Nguyen Quynh A. Le Takayuki Hirano Tatsuya Takemoto Michiko Nakai Dai‐ichiro Fuchimoto Takeshige Otoi 《Animal Science Journal》2019,90(1):55-61
Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system. 相似文献
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The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle. 相似文献
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Chi H Shinohara M Yokomine T Sato M Takao S Yoshida M Miyoshi K 《The Journal of reproduction and development》2012,58(1):69-76
RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation. 相似文献
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Zinc-finger nucleases (ZFNs) are engineered site-specific DNA cleavage enzymes that may be designed to recognize long target sites and thus cut DNA with high specificity. ZFNs mediate permanent and targeted genetic alteration via induction of a double-strand break at a specific genomic site. Compared to conventional homology-based gene targeting, ZFNs can increase the targeting rate by up to 100000-fold; gene disruption via mutagenic DNA repair is similarly efficient. The utility of ZFNs has been shown in many organisms, including insects, amphibians, plants, nematodes, and several mammals, including humans. This broad range of tractable species renders ZFNs a useful tool for improving the understanding of complex physiological systems, to produce transgenic animals, cell lines, and plants, and to treat human disease. This review summarized recent advances in the understanding of role and function of ZFNs and described the utility of ZFNs for mammalian transgenesis. 相似文献
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The inbred SLA miniature pig is a unique animal model developed for organ transplantation studies and pre‐clinical experimental purposes. Reported oestrous synchronization and superovulation treatments were examined in two SLA haplotypes (AA and DD) to allow collection of embryos for both practical embryo transfer and experimental technologies from a closed breeding colony. Pre‐puberal miniature pigs were poor responders to oestrous synchronization treatments, while post‐puberal sows were equivalent to commercial sows. Following superovulation, the ovulation number (corpora .hemorrhagica) was higher (p < 0.05) in the cycling sows when compared with non‐cycling sows. Ovulations were equivalent to commercial pre‐puberal gilts and non‐cycling sows (p > 0.05). No difference in ovulation number between haplotypes was observed, which differs from the previous report (DD>AA). Collection of zygotes for pronuclear injection was the highest in the non‐cycling post‐puberal miniature pig group (p < 0.05), although significantly lower when compared with the commercial pig treatment groups (p < 0.05). The incidence of cystic endometrial hyperplasia in our colony was equivalent to rates observed in commercial pigs. Pronuclear visualization following centrifugation was the highest in the non‐cycling miniature sow group and approximates to about 25% of ovulations and about half the rate observed in the commercial pigs (50%). Miniature pig embryos transferred between SLA haplotypes and transfer of DD embryos to commercial pigs resulted in live births at a higher efficiency than previously reported. This study demonstrates the feasibility of undertaking assisted reproductive technologies in a closed breeding colony of inbred SLA miniature pigs without compromise to the breeding programmes. 相似文献
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This retrospective study documents the first detection in Danish pigs of methicillin resistant and susceptible variants of the Staphylococcus aureus lineage ST398, which was previously described among pigs, pig farmers and veterinary staff in The Netherlands as well as in human patients and infected animals in Europe. Ten out of 100 pigs tested were shown to carry ST398 in the nasal cavity and two out of three farms tested were positive. The 10 porcine ST398 isolates had spa type t034 (n=9) and t1793 (n=1), and were resistant to erythromycin, clindamycin and tetracycline in addition to penicillin. The detection of ST398 in Danish pigs suggests that this new emerging zoonotic bacterium is rapidly spreading in the pig population in Europe. 相似文献
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This study was aimed to efficiently and accurately edit the growth trait-related gene insulin-like growth factor 2 (IGF2) in porcine fetal fibroblasts cells (PFFs) of Bama pigs using base editors.The IGF2 gene sequences of Bama pigs and Large White pigs were identified by PCR amplification and sequencing,and the expression vector of sgRNA-IGF2 targeting Bama pigs was constructed.Then the editing efficiency and product types at IGF2 gene via different base editors were studied by cell transfection,detection of mixed cell editing efficiency,screening of single cell colonies and genotyping.The results showed that there was a single nucleotide polymorphism (SNP) site at 3 072 bp in intron 3 of IGF2 gene between Bama pigs and Large White pigs,and a single-stranded oligonucleotide sequence targeting the IGF2 gene was designed.The single-stranded oligonucleotide was annealed and ligated with the BsaⅠ-linearized pGL3-U6-sgRNA plasmid to construct the sgRNA-IGF2 expression plasmid.The sequencing results of the recombinant plasmid showed that the sgRNA sequence was accurately inserted between the U6 promoter and the sgRNA scaffold.Four different cytosine base editors (rA1-BE3,hA3A-BE3,hA3A-BE3-Y130F and hA3A-eBE-Y130F) were co-transfected with sgRNA-IGF2-expressing plasmid into PFFs,respectively.The editing efficiency in mixed cells showed hA3A-BE3-based CBEs could introduce higher efficiency of C-to-T mutation than rA1-BE3 (P<0.05).The results of flow cytometry,single cell culture and genotyping showed that 71.43% of single cell colonies were mutant in hA3A-BE3 group,but 42.86% of them had insertions/deletions (indels).The editing efficiency of hA3A-BE3-Y130F (56.86%) and hA3A-eBE-Y130F (40.38%) were lower than hA3A-BE3(71.43%),but the indels of them were also lower (31.37% and 21.15%).In addition,single cell colonies with homozygous mutation at IGF2 gene site were achieved by base editors in this study.Base editors,as new gene editing tools,could efficiently and accurately modify SNP associated with economic traits in pig genome and accelerate genetic improvement. 相似文献
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本研究旨在利用碱基编辑器在巴马猪胎儿成纤维细胞（porcine fetal fibroblast cells,PFFs）中对生长性状相关基因胰岛素样生长因子2（insulin-like growth factor 2,IGF2）进行高效、精确的定点编辑。通过PCR扩增和测序对巴马猪和大白猪的IGF2基因序列进行鉴定,构建靶向巴马猪的sgRNA-IGF2表达载体,进而通过细胞转染、混合细胞编辑效率检测、单克隆细胞筛选及基因型鉴定等技术手段研究不同碱基编辑器对猪IGF2基因靶点的编辑效率及突变类型。结果显示,巴马猪和大白猪IGF2基因第3内含子存在第3 072 bp处的单核苷酸多态性（SNP）位点,设计靶向巴马猪IGF2基因的单链寡核苷酸序列。单链寡核苷酸经退火后与BsaⅠ线性化的pGL3-U6-sgRNA质粒进行重组连接,构建sgRNA-IGF2表达质粒,重组质粒测序结果表明,sgRNA序列已经精确连入U6启动子和sgRNA骨架之间。将rA1-BE3、hA3A-BE3、hA3A-BE3-Y130F和hA3A-eBE-Y130F 4种胞嘧啶碱基编辑器分别与sgRNA-IGF2表达质粒共转染至猪胎儿成纤维细胞中,对混合细胞编辑效率进行检测结果表明,对于IGF2基因靶点,hA3A-BE3系列碱基编辑器C到T的编辑效率显著高于rA1-BE3（P<0.05）。流式分选、单克隆细胞培养及鉴定结果表明,虽然有71.43%的hA3A-BE3单克隆细胞已被编辑,但有42.86%的细胞存在插入和缺失（indels）;hA3A-BE3-Y130F和hA3A-eBE-Y130F的编辑效率（56.86%和40.38%）虽低于hA3A-BE3（71.43%）,但是indels的效率也较低（31.37%和21.15%）。测序结果分析表明,利用碱基编辑器高效筛选到IGF2基因靶点纯合点突变的单克隆细胞。碱基编辑器作为新的基因编辑工具,可以对猪基因组中与经济性状关联的SNP位点进行高效、精确的基因修饰,为加速猪经济性状的遗传改良提供理论与实践基础。 相似文献