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1.
Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.  相似文献   

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锌指蛋白核酸酶的作用原理及其应用   总被引:1,自引:0,他引:1  
Zhong Q  Zhao SH 《遗传》2011,33(2):123-130
锌指蛋白核酸酶(Zinc finger nucleases,ZFN)因其能特异性识别并切割DNA序列以及可设计性,被用于基因定点突变和外源基因定点整合。目前,ZFN技术以其准确的靶位点设计能力和诱发高效率基因打靶的优势,越来越受到基因改造研究者的重视,已经成功应用于动植物细胞、胚胎的基因改造。随着鉴定靶DNA高亲和力的锌指蛋白(Zinc finger protein,ZFP)实验技术日渐成熟,可以预见到不久的将来这项技术会在基因工程和育种中得到广泛应用。文章介绍了锌指蛋白识别DNA靶位点和ZFN介导的基因打靶(Double strand break gene targeting,DSB-GT)的原理,同时还综述了目前ZFN技术用于基因改造的研究进展。  相似文献   

4.
Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ~1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.  相似文献   

5.
Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.  相似文献   

6.
Zinc finger nuclease (ZFN)-mediated gene targeting is rapidly becoming a powerful tool for "gene editing" and "directed mutagenesis" of plant and mammalian genomes including the human genome. ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically manipulate and permanently modify plant and mammalian genomes. Facile production of ZFNs and rapid characterization of their in vitro sequence-specific cleavage properties are a pre-requisite before ZFN-mediated gene targeting can become an efficient and effective practical tool for widespread use in biotechnology. Here, we report the design, engineering, and rapid in vitro characterization of ZFNs that target specific endogenous sequences within two mouse genes (mTYR and mCFTR), and two human genes (hCCR5 and hDMPK), respectively. These engineered ZFNs recognize their respective cognate DNA sites encoded in a plasmid substrate in a sequence-specific manner and, as expected, they induce a double-strand break at the chosen target site.  相似文献   

7.
Design, construction and in vitro testing of zinc finger nucleases   总被引:3,自引:0,他引:3  
Zinc finger nucleases (ZFNs) are hybrid proteins that have been developed as targetable cleavage reagents for double-stranded DNA, both in vitro and in vivo. This protocol describes the design and construction of new DNA-binding domains comprised of zinc fingers (ZFs) directed at selected DNA sequences. Because the ZFNs must dimerize to cut DNA, they are designed in pairs for any new site. The first step is choosing a DNA segment of interest and searching it for sequences that can be recognized by combinations of existing ZFs. The second step is the construction of coding sequences for the selected ZF sets. Third, these coding sequences are linked to that of the nonspecific cleavage domain from the FokI restriction endonuclease in a cloning vector of choice. Finally, the ZFNs are expressed in Escherichia coli, partially purified, and tested in vitro for cleavage of the target sequences to which they were designed. If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week.  相似文献   

8.
Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.  相似文献   

9.
Zinc finger nucleases (ZFNs) facilitate tailor-made genomic modifications in vivo through the creation of targeted double-stranded breaks. They have been employed to modify the genomes of plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double-stranded breaks at off-target sites. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites using Illumina sequencing. Only a minority of these off-target sites accumulated lesions, where the thermodynamics of zinc finger–DNA recognition appear to be a defining feature of active sites. Surprisingly, we observed that both the specificity of the incorporated zinc fingers and the choice of the engineered nuclease domain could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both zinc finger-dependent and -independent characteristics that can be tailored to create ZFNs with greater precision.  相似文献   

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The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.  相似文献   

12.
Dong Z  Ge J  Li K  Xu Z  Liang D  Li J  Li J  Jia W  Li Y  Dong X  Cao S  Wang X  Pan J  Zhao Q 《PloS one》2011,6(12):e28897
Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6)) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7)), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+) and 14 mstn(nju7/+) yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.  相似文献   

13.
设计表达了四个锌指核酸酶,用于切断人基因组中的rRNA基因家族的内部转录间隔序列,造成双链断裂,以此提高针对多位点基因打靶的效率,为后续基因打靶应用于基因治疗研究奠定基础。首先,在人rRNA基因家族ITS1序列中找到两个合适的9 bp长的序列(中间间隔6 bp)为锌指蛋白识别位点,根据识别位点序列每个位点分别设计两个三锌指蛋白。通过设计引物进行重叠延伸PCR得到全长编码锌指蛋白的DNA,分别克隆到表达载体pET-28a(+),构建重组质粒pET28a-ZFP,转化大肠杆菌RossettaTM(DE3),实现带组氨酸标签的锌指融合蛋白的表达与纯化。同时,将限制性内切酶Fok I的切割结构域分别与四个锌指蛋白序列采用PCR拼接后克隆到表达载体pET-28a(+),构建重组质粒pET28a-ZFN,转化到大肠杆菌RossettaTM(DE3),实现带组氨酸标签的锌指核酸酶融合蛋白的表达并纯化。  相似文献   

14.
Generation of transgenic cattle by lentiviral gene transfer into oocytes   总被引:14,自引:0,他引:14  
The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.  相似文献   

15.
The therapeutic efficacy of adoptively transferred cytotoxic T lymphocytes (CTL) has been demonstrated in clinical trials for the treatment of chronic myelogenous leukemia, cytomegalovirus-mediated disease, and Epstein-Barr virus-positive B cell lymphomas. It is however limited by the difficulty of generating sufficient amounts of CTLs in vitro, especially for the treatment of solid tumors. Recent gene therapy approaches, including two clinical trials, successfully apply genetic engineering of T cell specificity by T cell receptor (TCR) gene transfer. In this review we want to elucidate several principles of the redirection of T cell specificity. We cover basic aspects of retroviral gene transfer, regarding transduction efficacy and transgene expression levels. It was demonstrated that the number of TCR molecules on a T cell is important for its function. Therefore, an efficient transfer system that yields high transduction efficiency and strong and stable transgene expression is a prerequisite to achieve effector function by redirected T cells. Furthermore, we consider more recent aspects of T cell specificity engineering. These include the possibility of co-transferring coreceptors to create for example functional T helper cells by engrafting CD4(+) T cells with a MHC class I restricted TCR and the CD8 coreceptor and vice versa. Also, risks related to the adoptive transfer of TCR gene-modified T cells and possible safety mechanisms are discussed. Finally, we summarize recent findings describing transferred TCRs capable of displacing endogenous TCRs from the cell surface.  相似文献   

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Placental dysfunction underlies many complications during pregnancy, and better understanding of gene function during placentation could have considerable clinical relevance. However, the lack of a facile method for placenta-specific gene manipulation has hampered investigation of placental organogenesis and the treatment of placental dysfunction. We showed previously that transduction of fertilized mouse eggs with lentiviral vectors leads to transgene expression in both the fetus and the placenta. Here we report placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts after removal of the zona pellucida. All of the placentas analyzed, but none of the fetuses, were transgenic. Application of this method substantially rescued mice deficient in Ets2, Mapk14 (also known as p38alpha) and Mapk1 (also known as Erk2) from embryonic lethality caused by placental defects. Ectopic expression of Mapk11 also complemented Mapk14 deficiency during placentation.  相似文献   

18.
Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay. The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.  相似文献   

19.
Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.  相似文献   

20.
Lentivirus‐derived vectors (LVs) were studied for the generation of stable recombinant Chinese hamster ovary (CHO) cell lines. Stable pools and clones expressing the enhanced green fluorescent protein (eGFP) were selected via fluorescence‐activated cell sorting (FACS). For comparison, cell pools and cell lines were also generated by transfection, using the LV transfer plasmid alone. The level and stability of eGFP expression was greater in LV‐transduced cell lines and pools than in those established by transfection. CHO cells were also infected at two different multiplicities of infection with an LV co‐expressing eGFP and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). At 2‐day post‐infection, clonal cell lines with high eGFP‐specific fluorescence were recovered by FACS. These clones co‐expressed TNFR:Fc with yields of 50–250 mg/L in 4‐day cultures. The recovered cell lines maintained stable expression over 3 months in serum‐free suspension culture without selection. In conclusion, LV‐mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high‐producing recombinant cell lines. Biotechnol. Bioeng. 2011; 108:600–610. © 2010 Wiley Periodicals, Inc.  相似文献   

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