Generation and identification of endothelial-specific Hrh2 knockout mice
Rui MengWen-Ke CaiWenmang XuQiang FengPing WangYanhua HuangYuxin FanTao ZhouQin YangZhi‐Ran LiGong‐Hao He
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Knockout mouse
Gene targeting
Chimera (genetics)
Gene knockout
Gene knockin
Conditional gene knockout
Generation of specific gene knockout mouse model is a reliable technique for studying the role of genes in mammalian model organism.One of the key steps to acquire a gene knockout mouse is to construct a targeting vector for homologous recombination in mouse embryonic stem cells.For some genes,the mutants will die in uteri owing to their critical roles in embryonic development,or the mutant mice may have different phenotypes according to the period of development and types of tissues.It is difficult to study these genes by the conventional knockout approaches.Thereby the conditional knockout strategy had been developed for its advantages to circumvent the embryonic lethality problem and to investigate gene function temporally and spatially.Using modified Red homologous recombineering system,the two LoxP sites were inserted into the target position accurately and the GPR126 conditional gene-targeting vector was rapidly constructed.The successful generation of GPR126 conditional knockout construct will be helpful for the subsequent production of knockout mouse model and exploration of the function of GPR126 in mice.
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Inducible conditional knockout mice are important tools for studying gene function and disease therapy, but their generation is costly and time-consuming. We introduced clustered regularly interspaced short palindromic repeats (CRISPR) and Cre into an LSL-Cas9 transgene-carrying mouse line by using adeno-associated virus (AAV)-PHP.eB to rapidly knockout gene(s) specifically in central nervous system (CNS) cells of adult mice. NeuN in neurons and GFAP in astrocytes were knocked out 2 weeks after an intravenous injection of vector, with an efficiency comparable to that of inducible Cre-loxP conditional knockout. For functional testing, we generated astrocyte-specific Act1 knockout mice, which exhibited a phenotype similar to mice with Cre-loxP-mediated Act1 knockout, in an animal model of multiple sclerosis (MS), an autoimmune disorder of the CNS. With this novel technique, neural cell-specific knockout can be induced rapidly (few weeks) and cost-effectively. Our study provides a new approach to building inducible conditional knockout mice, which would greatly facilitate research on CNS biology and disease. Inducible conditional knockout mice are important tools for studying gene function and disease therapy, but their generation is costly and time-consuming. We introduced clustered regularly interspaced short palindromic repeats (CRISPR) and Cre into an LSL-Cas9 transgene-carrying mouse line by using adeno-associated virus (AAV)-PHP.eB to rapidly knockout gene(s) specifically in central nervous system (CNS) cells of adult mice. NeuN in neurons and GFAP in astrocytes were knocked out 2 weeks after an intravenous injection of vector, with an efficiency comparable to that of inducible Cre-loxP conditional knockout. For functional testing, we generated astrocyte-specific Act1 knockout mice, which exhibited a phenotype similar to mice with Cre-loxP-mediated Act1 knockout, in an animal model of multiple sclerosis (MS), an autoimmune disorder of the CNS. With this novel technique, neural cell-specific knockout can be induced rapidly (few weeks) and cost-effectively. Our study provides a new approach to building inducible conditional knockout mice, which would greatly facilitate research on CNS biology and disease.
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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.
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In the last decade, gene targeting in embryonic stem (ES) cells has been extensively used as a powerful tool to study gene function in the mouse, as a mammalian model organism. As initially developed, the technique allows the disruption of a target gene in the murine germline by the insertion of a selectable marker (). The vast majority of the more than 1000 knockout mice in existence have been created following this design. Many of these strains have given valuable information on the biologic function of the genes studied (). Since these "conventional" knockout mice are usually homozygous for a null allele in the germline, they provide an appropriate model for inherited diseases, leading to embryonic or early postnatal lethality in about 30% of cases. Apart from this application, germline knockout mice do not necessarily represent the best technical approach for studying other aspects of gene function in vivo, in particular in adult mice. A refined knockout strategy termed conditional gene targeting has been developed () that permits the inactivation of the target gene to be restricted to a certain organ and/or developmental stage (). Figure 1 depicts the principal difference between the two strategies, comparing a germline knockout mouse with two types of conditional mutants in which the target becomes inactivated either early on in a particular organ without temporal control or upon induction at a chosen time point. The inactivation of the target gene in a conditional mutant is achieved by the expression of a site-specific DNA recombinase (Cre or FLP) in mice in conjunction with the introduction of two recombinase recognition sequences [loxP or FLP recognition target (FRT)] into noncoding regions of the target gene (Fig. 2). These sites are usually placed in the same orientation into introns such that recombination results in gene inactivation through the deletion of the loxP- or FRT-flanked exon(s). Although the generation of completely ES cell-derived mice has been recently significantly improved (), the current standard method to derive a conditional mutant requires the generation of two mouse strains: one mouse strain harboring a loxP- or FRT-flanked gene segment by gene targeting in ES cells and a second transgenic strain expressing Cre or FLP either constitutively, or upon induction, in one or several organs. The conditional mutant is generated by crossing these two strains such that the inactivation of the target gene will be restricted in a spatial and temporal manner, following the pattern of Cre or FLP expression in the transgenic strain (Fig. 2, right side). As the homozygous loxP- or FRT-flanked allele must be combined with a heterozygous recombinase transgene, it often requires additional generation time (3 months) to obtain reasonable numbers of conditional mutants, compared with germline KO mice. The loxP-containing strain can be also converted into a null allele mutant by a single cross to a deleter strain expressing Cre in germ cells or the early embryo (Fig. 2, left side). There are no general rules to decide whether the germline or the conditional mutation is more appropriate for a particular experiment since this depends on the biologic question. In fact, both types of mutants are usually generated at the same time to investigate gene function both during embryonic development and in the adult animal. However, following the conditional mutagenesis scheme described in Subheading 1.4., germline and conditional mutants can be generated for a particular gene using a single targeted ES cell clone. Thus, we would recommend the conditional gene targeting scheme for all knockout projects, as this offers a greater flexibility compared with the conventional approach without additional efforts once the necessary reagents are assembled. Open image in new window Fig. 1. Gene-targeting strategies. Upper row: the knockout (KO) is transmitted through the germline, resulting in a null allele mutant strain (conventional KO); middle row: the KO is introduced in somatic cells and restricted to a specific tissue (conditional, cell type-specific KO); bottom row: the KO is introduced upon induction (conditional, inducible KO). Tissues in which the target gene is inactivated are shown in black. Open image in new window Fig. 2. Generation of a conditional mutant. Upper mouse: a strain harboring a loxP-flanked (triangles) gene segment (square). To derive a conditional mutant, the strain is crossed to mice expressing Cre recombinase constitutively or upon induction in specific cell types or organs (right side). Gene modification occurs by Cre-mediated recombination according to the expression pattern of the recombinase transgene. The loxP-containing strain can be converted into a null allele mutant by a single cross to a deleter strain expressing Cre in germ cells or the early embryo (left side).
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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。.
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The directed introduction of null mutations into defined genes has proven invaluable in elucidating gene function in a variety of experimental organisms. In the last decade or so this approach has been extended to mice (1) by the combined use of homologous recombination in murine embryonic stem (ES) cells to precisely target a mutation to a desired gene and subsequent derivation of mice carrying the targeted gene alteration from the genetically manipulated ES cells (e.g., by injection of gene- modified ES cells into blastocysts with subsequent germline transmission). In most instances null, or knockout (KO), mutations have been generated in mice by either simple insertion of a neo selectable marker in the target gene or neo insertion coupled with deletion of a critical region of the target gene. Targeted null mutations in a gene of interest, however, can lead to embryonic lethality in mice, thus obscuring the particular role of that gene in a target tissue or in the adult.
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KCNQ1 is a voltage-activated potassium channel α -subunit expressed in various cell types, including cardiac myocytes and epithelial cells. KCNQ1 associates with different β-subunits of the KCNE protein family. In the human heart, KCNQ1 associates with KCNE1 to generate the I Ks current characterized by its slow activation and deactivation kinetics. Mutations in either KCNQ1 or KCNE1 are responsible for at least four channelopathies that lead to cardiac dysfunction and one that leads to congenital deaf-ness: the Romano-Ward syndrome, the short QT syndrome, atrial fibrillation, and the Jervell and Lange-Nielsen syndrome (cardioauditory syndrome). To date, nearly 100 different KCNQ1 mutations have been reported as responsible for the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. Patch clamp and immunofluorescence techniques are instrumental for characterization of the molecular mechanisms responsible for the altered function of KCNQ1 and its partners.
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Diseases with a genetic basis can be modeled with knockout, knock-in, and conditional mutant gene-targeted mice. In the following, we provide detailed protocols for gene targeting. Gene targeting of embryonic stem cells can be accomplished by laboratories equipped for tissue culture. Alternatively, many gene-targeting services divide the work of targeting with a customer lab. In this collaborative situation, knowledge of the entire process helps ensure a successful outcome. The construction of chimeras for germ-line transmission is not described here, because this procedure is beyond the means of most laboratories, typically is provided by transgenic core facilities, and is best learned through hands-on demonstration.
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