One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting

The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout(CKO) models in zebrafish due to the low efficiency of homologous recombination(HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin(KI) switch(zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2 ~(zCKOIS)which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2 ~(zCKOIS)was switched to a non-functional CKO allele hey2 ~(zCKOIS)-inv associated with Tag RFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our z CKOIS strategy an applicable CKO approach for zebrafish.     

Genetic knockouts and knockins in human somatic cells

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.     

Efficient somatic gene targeting in the lymphoid human cell line DG75

Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.     

Efficient inducible Pan-neuronal cre-mediated recombination in SLICK-H transgenic mice

Large-scale functional genomics in mice is becoming feasible through projects to develop conditional knockout alleles for every gene. Inducible neuron-specific gene knockout in such mice will permit the analysis of neuronal phenotypes while circumventing developmental defects or embryonic lethality. Here we describe a transgenic line, termed SLICK-H, that facilitates widespread inducible conditional genetic manipulation within most populations of projection neurons. In SLICK-H mice, the Thy1 promoter drives robust and relatively uniform expression of a drug-inducible form of cre recombinase throughout the peripheral and central nervous system. This permits efficient induction of cre-mediated genetic manipulation upon tamoxifen administration in adult mice. Importantly, cre activity in the absence of tamoxifen is minimal, permitting tight control of recombination. In the present study, we catalog in detail the transgene expression patterns and recombination efficiencies in SLICK-H mice. Our results highlight the utility of SLICK-H mice for functional genomics in the nervous system.     

Construction of Gene-Targeting Vectors: a Rapid Mu in vitro DNA Transposition-Based Strategy Generating Null,Potentially Hypomorphic,and Conditional Alleles

Gene targeting into mammalian genomes by means of homologous recombination is a powerful technique for analyzing gene function through generation of transgenic animals. Hundreds of mouse strains carrying targeted alleles have already been created and recent modifications of the technology, in particular generation of conditional alleles, have extended the usefulness of the methodology for a variety of special purposes. Even though the standard protocols, including the construction of gene-targeting vector plasmids, are relatively straightforward, they typically involve time-consuming and laborious gene mapping and/or sequencing steps. To produce various types of gene-targeting constructions rapidly and with minimum effort, we developed a strategy, that utilizes a highly efficient in vitro transposition reaction of phage Mu, and tested it in a targeting of the mouse Kcc2 gene locus. A vast number and different types of targeting constructions can be generated simultaneously with little or no prior sequence knowledge of the gene locus of interest. This quick and efficient general strategy will facilitate easy generation of null, potentially hypomorphic, and conditional alleles. Especially useful it will be in the cases when effects of several exons within a given gene are to be studied, a task that necessarily will involve generation of multiple constructions. The strategy extends the use of diverse recombination reactions for advanced genome engineering and complements existing recombination-based approaches for generation of gene-targeting constructions.     

Generation of an EphA4 conditional allele in mice

Ephrins and Eph receptor tyrosine kinases are cell‐surface molecules that serve a multitude of functions in cell–cell communication in development, physiology, and disease. EphA4 is a promiscuous member of the EphA subclass of Eph receptors and can bind to both EphrinAs and EphrinBs. In addition to its well‐established roles in guiding the development of neuronal connectivity, EphA4 has been implicated for a role in synaptic plasticity, vascular formation, axon regeneration, and central nervous system repair following injury. However, the study of its role in the adult stage has been hampered by confounding developmental defects in EphA4 germline mutants. Here, we report the generation and molecular characterization of an EphA4 conditional allele along with a novel null allele with a knockin fluorescent reporter gene (mCFP). The conditional allele will be useful in ascertaining postdevelopmental and/or cell type‐specific function of EphA4 in physiology, injury, and disease. genesis 48:101–105, 2010. © 2009 Wiley‐Liss, Inc.     

Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology

Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx‐based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene‐trap mutagenesis. The SAGFLEx gene‐trap cassette comprises the rabbit β‐globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site‐specific recombinases Cre and Flp. Insertion of the gene‐trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene‐trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial‐ and temporal‐controlled manner. genesis 54:19–28, 2016. © 2015 Wiley Periodicals, Inc.     

Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.     

Generation of conditional knockout alleles for PDGF-C

PDGF-C is a newly identified member of the platelet-derived growth factor (PDGF) family, which is involved in multiple cellular functions by signaling through PDGF receptor (PDGFR)-alphaalpha and alphabeta dimers. PDGF-C deficiency is perinatal lethal due to the formation of cleft palate. To further characterize the cellular function of PDGF-C during both embryonic and postnatal development, we have generated two conditional alleles of the Pdgf-c gene in which two loxP sites flank exon 5. Global Cre-mediated excision of the floxed exon 5 in these alleles resulted in a complete loss of PDGF-C expression and caused embryonic defects identical to those previously described for the PDGF-C null embryos. These conditional alleles will therefore be the important genetic tools for dissecting the spatial and temporal roles of PDGF-C during development and in adult tissues. Furthermore, from this work, we have also described a simple approach for creating mouse conditional alleles in an efficient manner.     

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7×10⁻⁶. In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F₁ generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P₀ generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism. ^★Equal contributions to this article.     

Production of conditional point mutant knockin mice

Genetically engineered mice with point mutations in endogenous genes (i.e., knockin mice) are extremely useful tools for dissecting gene function. Currently available methodologies for creating knockin mice are limited in that the introduced mutation is globally present in all cells of the animal from conception through adulthood. In this report, we describe a strategy for creating mice in which a point mutant allele replaces the wild type allele in a conditional manner, e.g., in a tissue-specific and/or temporally restricted pattern. As proof of concept, we created mice that conditionally harbor a point mutated gamma-aminobutyric acid receptor subunit. In the absence of Cre recombinase, the engineered allele produces only wild type product with no evidence of expression of the mutant. In contrast, following Cre-mediated recombination, only the point mutant product is produced. By restricting Cre expression to subpopulations of neurons of postnatal animals, we demonstrate tissue-specific regulation of the point mutant knockin. This strategy will be useful for a wide variety of studies that require precise conditional replacement of an endogenous wild type gene with a point mutant.     

Tamoxifen feeding method is suitable for efficient conditional knockout

The Cre-driver system is used to generate conditional knockout mice. Tamoxifen inducible Cre-driver mice can be used for spatiotemporal knockout by administration of the drug. A major tamoxifen administration is performed by intraperitoneal administration or oral administration. However, these forced administrations may be damaging to mice. Herein, we have demonstrated an improved method of administering tamoxifen with powdered food to mice. A mouse line expressing the tamoxifen-inducible Cre gene was used ubiquitously in this experiment to evaluate the efficiency of Cre recombination in the whole body. Our method also achieved efficient recombination without causing injury to mice. The X-gal staining intensity of the feeding method was equivalent to that of the intraperitoneal administration method. Furthermore, this method can be used for recombination before birth, or during the fetal period. We recommend researchers to employ this feeding method to administer tamoxifen to minimize the risk of injury to mice.     

Generation of mice with conditional null allele for GdX/Ubl4A

GdX (also named Ubl4A) is a house-keeping gene located on the X chromosome and encodes a protein harboring an ubiquitin-like domain in human and mouse. Although identified in 1988, the function of GdX remains unknown. To elucidate the role of GdX in vivo, we generated a conditional GdX knockout mouse in which Exon 2 was flanked by two loxP sites. We obtained viable and fertile mice with homozygous GdX(flox/flox) or GdX(flox/Y) allele. Germ-line transmission was confirmed by crossing the mouse bearing conditionally targeted allele with an EIIα-Cre transgenic mouse. GdX was successfully depleted in tissues of EIIα-Cre-GdX-null mice. GdX(-/-) and GdX(-/Y) mice are viable and exhibit normal development compared with wild-type littermates within 6 months during our observation. We also observed that GdX knockout male mice were functionally normal in the reproductive system where Ubl4B was specifically expressed. GdX(flox/flox) and GdX(flox/Y) conditional mice provide a tool for further tissue-specific function analysis of the GdX protein under different conditions.     

Improving the efficiency for generation of genome-edited zebrafish by labeling primordial germ cells

Although CRISPR/Cas, a new versatile genome-editing tool, has been widely used in a variety of species including zebrafish, an important vertebrate model animal for biomedical research, the low efficiency of germline transmission of induced mutations and particularly knockin alleles made subsequent screening for heritable offspring tedious, time-consuming, expensive and at times impossible. In this study, we reported a method for improving the efficiency of germline transmission screening for generation of genome-edited zebrafish mutants. Co-microinjecting yfp-nanos3 mRNA with Cas9 mRNA, sgRNA and single strand DNA donor to label the distribution of microinjected nucleotides in PGCs (primordial germ cells), we demonstrated that founders carrying labeled PGCs produced much higher numbers of knockin and knockout progeny. In comparison with the common practice of selecting founders by genotyping fin clips, our new strategy of selecting founders with tentatively fluorescent-labeled PGCs significantly increase the ease and speed of generating heritable knocking and knockout animals with CRISPR/Cas9.     

A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles

Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.     

High throughput gene trapping and postinsertional modifications of gene trap alleles

Gene trapping is a high-throughput insertional mutagenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, gene trapping helped to generate tenth of thousands of ESC lines harboring mutations in single genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout Mouse Consortium (IKMC; www.knockoutmouse.org) aim to generate conditional alleles for every protein coding gene in the mouse genome by high throughput conditional gene targeting and trapping. Here, we provide protocols for gene trapping in ESCs that can be easily adapted to any other mammalian cell. We further provide protocols for handling and verifying conditional gene trap alleles in ESC lines obtained from the IKMC repositories and describe a highly efficient method for the postinsertional modification of gene trap alleles. More specifically, we describe a protein tagging strategy based on recombinase mediated cassette exchange (RMCE) that enables protein localization and protein-protein interaction studies under physiological conditions.     

标准化嵌合体制备体系的研究

目的:完善和规范基因剔除小鼠技术体系的关键技术环节,建立一套高效的嵌合体制备体系。方法:优化胚胎干细胞(ES细胞)的基本培养条件;应用条件培养液筛选、富集高嵌合潜能的ES细胞;成熟嵌合体的制备技术;改变胚胎的移植方式,改善受体的生理状态。结果:ES细胞基本培养条件的优化及条件培养液的筛选保持了ES细胞的整体高嵌合潜能,嵌合体制备技术得以成熟,胚胎移植方式的改变提高了移植胚胎的产仔率,这些措施大大提高了嵌合体的制备效率。结论:通过对基因剔除小鼠技术体系的关键技术环节进行优化和改进,建立了一套高效的嵌合体制备程序,为基因剔除小鼠服务体系的开展打下了坚实的基础。