[Progress in gene knockout mice].
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Abstract:
The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.基因敲除小鼠技术的建立和发展使得人们为研究基因的功能和寻找新的治疗人类疾病的靶点提供了强有力的支持。基因打靶和基因捕获是两种通过胚胎干细胞 (Embryonic stem cell，ESC) 构建基因敲除小鼠的技术。基因打靶通过同源重组替换内源基因从而敲除目的基因，而基因捕获则有启动子捕获和polyA 捕获两种方法对目的基因进行敲除。近年来，有许多新的基因敲除技术不断被开发出来，包括Cre/loxP 系统、CRISP/Cas9系统以及最新的ZFN 技术和TAILEN 技术，都有望取代传统基因敲除手段。文中简要阐述了如今新出现的几种基因敲除小鼠技术。.Keywords:
Gene knockout
Gene targeting
Zinc finger nuclease
Knockout mouse
Gene knockin
Conditional gene knockout
Cre-Lox recombination
ABSTRACT Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here we present a simple method that combines the use of CRISPR-Cas9 and Cre-loxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next CRISPR-Cas9 is used to knockout an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate potential mutants of the tested gene.
Cre recombinase
Gene knockout
Conditional gene knockout
Gene knockin
Cre-Lox recombination
Gene targeting
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The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.基因敲除小鼠技术的建立和发展使得人们为研究基因的功能和寻找新的治疗人类疾病的靶点提供了强有力的支持。基因打靶和基因捕获是两种通过胚胎干细胞 (Embryonic stem cell，ESC) 构建基因敲除小鼠的技术。基因打靶通过同源重组替换内源基因从而敲除目的基因，而基因捕获则有启动子捕获和polyA 捕获两种方法对目的基因进行敲除。近年来，有许多新的基因敲除技术不断被开发出来，包括Cre/loxP 系统、CRISP/Cas9系统以及最新的ZFN 技术和TAILEN 技术，都有望取代传统基因敲除手段。文中简要阐述了如今新出现的几种基因敲除小鼠技术。.
Gene knockout
Gene targeting
Zinc finger nuclease
Knockout mouse
Gene knockin
Conditional gene knockout
Cre-Lox recombination
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Abstract The establishment of mouse embryonic stem (ES) cell lines has allowed for the gene?ration of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene‐targeting experiment. This unit describes detailed step‐by‐step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. Curr. Protoc. Cell Biol . 44:19.13.1‐19.13.24. © 2009 by John Wiley & Sons, Inc.
Gene knockout
Knockout mouse
Gene targeting
Conditional gene knockout
Gene knockin
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Technique of homologous recombination based gene targeting developed in the late 1980s and won the Nobel Prize in Physiology or Medicine in 2007. However, this technique could only performed in mice which embryonic stem (ES) cells could keep in the potential of multifunction in vitro. Therefore gene knockout technology was difficult to be applied in other species of animals for a long time. Since 2008, with the development of the new technologies, such as the new ES cell gene targeting, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clusters of regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9), building gene knockout in large and medium animals models which are similar to human in disease research becomes possible. This review describes some new gene knockout technologies in large and medium animal models for recent years.
Gene knockout
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Objective
To explore for the construction of microRNA-155 (miR-155) gene knockout mouse model by using CRISPR/Cas9 technique.
Methods
Healthy C57BL/6J mice were selected. First, the target sequence of mouse genome was amplified and sequenced to identify the miR-155 sequence and loci. Then the Cas9 vector (Cas9 RNA) and targeting vector single guide RNA (sgRNA) were designed and constructed. After in-vitro transcription, Cas9 RNA and sgRNA were injected into the mouse fertilized eggs which were cultured in vitro. The qualified embryos were transplanted into the fallopian tubes of the surrogate mice which then gave birth to the F0 generation mice. The gene knockout of F0 generation mice was examined by the genotyping. Thereby, knockout mice model was established and bred.
Results
Eighteen mice of F0 generation were obtained by CRISPR/Cas9 technique. Mouse No.4 presented with deleted base segments in single-stranded DNA and stably passagable genotype. After reproduction, three F1-generation heterozygous mice were obtained. The heterozygous mice were mated to obtain F2 homozygous knockout mice. Electrophoresis and gene-sequencing confirmed deletion of 114-bp base sequence in the off-springs, suggesting successful knockout of miR-155 gene. The miR-155 gene knockout mice could reproduce normally in clean-grade animal breeding environments.
Conclusion
CRISPR/Cas9 technique can be used for quick and effective construction of miR-155 gene knockout mice models.
Key words:
MicroRNA-155; CRISPR/Cas9; Models, animal; Mice; Gene knockout
Gene knockout
Knockout mouse
Gene targeting
Conditional gene knockout
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The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.
Conditional gene knockout
Cre recombinase
Gene knockout
Gene targeting
Cre-Lox recombination
Gene knockin
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Gene knockout
Conditional gene knockout
Gene knockin
Cre-Lox recombination
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Abstract The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.
Conditional gene knockout
Gene knockout
Gene knockin
Knockout mouse
Cre recombinase
Cre-Lox recombination
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Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here, we present a simple method that combines the use of CRISPR-Cas9 and Cre-LoxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next, CRISPR-Cas9 is used to knock out an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein, enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate the ability of phylogenic homologues and of potential mutants to cover functionally for the deleted gene.
Cre recombinase
Gene knockout
Gene knockin
Cre-Lox recombination
Conditional gene knockout
Gene targeting
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Zinc finger nuclease
Gene knockout
Genome Engineering
Gene targeting
Gene knockin
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