Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.     

Contemporary gene targeting strategies for the novice

Gene targeting in mouse embryonic stem (ES) cells is a fundamental methodology for generating mice with precise genetic modifications. Although there are many complex gene targeting strategies for creating a variety of diverse mutations in mice, most investigators initially choose to generate a null allele. Here we provide a guide for the novice to generate a null allele for a protein coding gene using a fundamental gene targeting strategy. Ultimately, a well considered gene targeting strategy saves significant amounts of time, money, and research animal lives. The straightforward strategy presented here bypasses many of the pitfalls associated with gene knockouts generated by novices. This guide also serves as a foundation for subsequently designing more complex gene targeting strategies.     

A morpholino phenocopy of the cyclops mutation

Summary: Conditional and tissue specific gene targeting using the Cre‐loxP recombination system in combination with established ES cell techniques has become a standard for in vivo loss of function studies. In a typical flox and delete gene targeting strategy, the loxP‐neo‐loxP cassette is inserted into an intron and an additional loxP site is located in one of the homology arms so that loxP sites surround a functionally essential part of the gene. The neo cassette in usually removed by transient expression of the Cre recombinase in ES cells to avoid selection gene interference and genetic ambiquity. However, this causes a significant increase in manipulation of ES cells and often compromises ES cell pluripotency. Here we describe a method in which the floxed neo gene is removed from a knockout allele by infecting 16‐cell‐stage morulae by the recombinant Cre adenovirus. This virus provides only transient Cre expression and does not integrate into the mouse genome. Produced mosaic mice transmitted the desired allele without the neo cassette with high frequency to their offspring. This method is rapid and easy and does not require any special equipment. Moreover, because superovulated mice can be used as donors, this method does not necessitate a large number of mice. genesis 31:126–129, 2001. © 2001 Wiley‐Liss, Inc.     

Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system

Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2^loxP/loxP mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.     

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.     

Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem(ES)cells,which is used to produce gene-targeted mice for wide applications in biomedicine.However,a major bottleneck in this approach is that the robustness of germiine transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing,which impairs the efficiency and robustness of mouse model generation.Recently,we have established a new type of pluripotent cells termed extended pluripotent stem(EPS)cells,which have superior developmental potency and robust germline competence compared to conventional mouse ES cells.In this study,we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage.Based on gene targeting in mouse EPS cells,we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation,Haibo Li and Chaoran Zhao contributed equally to this work.Electronic supplementary material The online version of this article(https://doi.org/10.1007/s13238-018-0556-1)contains supplementary material,which is available to authorized users.which could be accomplished in approximately 2 months.Importantly,using this approach,we successfully constructed mouse models in which the human interleukin 3(IL3)or interleukin 6(IL6)gene was knocked into its corresponding locus in the mouse genome.Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting,which have great application potential in biomedical research.     

A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ

A Cre recombinase expression cassette was inserted into the X-linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this "Cre-deleter" transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene.     

Targeted integration in rat and mouse embryos with zinc-finger nucleases

Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell-based targeting technology, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat and the mouse by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.     

From mouse to man: generating megabase chromosome rearrangements

Experimental approaches for deciphering the function of human genes rely heavily on our ability to generate mutations in model organisms such as the mouse. However, because recessive mutations are masked by the wild-type allele in the diploid context, conventional mutagenesis and screening is often laborious and costly. Chromosome engineering combines the power of gene targeting in embryonic stem (ES) cells with Cre--loxP technology to create mice that are functionally haploid in discrete portions of the genome. Chromosome deletions, duplications and inversions can be tagged with visible markers, facilitating strain maintenance. These approaches allow for more refined mutagenesis screens that will greatly accelerate functional mouse genomics and generate mammalian models for developmental processes and cancer.     

Gene trap and gene inversion methods for conditional gene inactivation in the mouse

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.     

Identification of germline competent chimaeras by copulatory plug genotyping

The increasing use of ES cell lines from strains other than 129, in particular C57BL/6, has greatly reduced the time taken to generate gene knockouts on a defined genetic background. Generally, C57BL/6 ES cell lines transmit less efficiently through the germline than 129 lines; consequently the burden on animal husbandry at this stage is increased. Genotyping sperm from chimaeric males may be used to identify mice which are transmitting the manipulated allele, however it requires that the mice be culled and the sperm used for IVF. Here we describe a quick and reliable method for genotyping copulatory plugs. Males which produce a positive result can then be naturally mated. Thus far we have observed a perfect correlation between copulatory plug genotype and germline transmission, accompanied by considerable savings in mouse numbers and resources.     

Recycling selectable markers in mouse embryonic stem cells.

As a result of gene targeting, selectable markers are usually permanently introduced into the mammalian genome. Multiple gene targeting events in the same cell line can therefore exhaust the pool of markers available and limit subsequent manipulations or genetic analysis. In this study, we describe the combined use of homologous and CRE-loxP-mediated recombination to generate mouse embryonic stem cell lines carrying up to four targeted mutations and devoid of exogenous selectable markers. A cassette that contains both positive and negative selectable markers flanked by loxP sites, rendering it excisable by the CRE protein, was constructed. Homologous recombination and positive selection were used to disrupt the Rep-3 locus, a gene homologous to members of the mutS family of DNA mismatch repair genes. CRE-loxP-mediated recombination and negative selection were then used to recover clones in which the cassette had been excised. The remaining allele of Rep-3 was then subjected to a second round of targeting and excision with the same construct to generate homozygous, marker-free cell lines. Subsequently, both alleles of mMsh2, another mutS homolog, were disrupted in the same fashion to obtain cell lines homozygous for targeted mutations at both the Rep-3 and mMsh2 loci and devoid of selectable markers. Thus, embryonic stem cell lines obtained in this fashion are suitable for further manipulation and analysis involving the use of selectable markers.     

Targeted integration of DNA using mutant lox sites in embryonic stem cells.

Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albertet al. (1995)Plant J., 7, 649-659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type lox Por LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression. Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutatedloxsystem will be useful for 'knock-in' integration of DNA in ES cells.     

EUCOMM--the European conditional mouse mutagenesis program.

Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.     

Introducing defined chromosomal rearrangements into the mouse genome

Chromosomal rearrangements have been instrumental in genetic studies in Drosophila. Visibly marked deficiencies (deletions) are used in mapping studies and region-specific mutagenesis screens by providing segmental haploidy required to uncover recessive mutations. Marked recessive lethal inversions are used as balancer chromosomes to maintain recessive lethal mutations and to maintain the integrity of mutagenized chromosomes. In mice, studies on series of radiation-induced deletions that surround several visible mutations have yielded invaluable functional genomic information in the regions analyzed. However, most regions of the mouse genome are not accessible to such analyses due to a lack of marked chromosomal rearrangements. Here we describe a method to generate defined chromosomal rearrangements using the Cre--loxP recombination system based on a published strategy [R. Ramirez-Solis, P. Liu, and A. Bradley, (1995) Nature 378, 720--724]. Various types of rearrangements, such as deletions, duplications, inversions, and translocations, can be engineered using this strategy. Furthermore, the rearrangements can be visibly marked with coat color genes, providing essential reagents for large-scale recessive genetic screens in the mouse. The ability to generate marked chromosomal rearrangements will help to elevate the level of manipulative mouse genetics to that of Drosophila genetics.     

Cre recombinase activity is inhibited in vivo but not ex vivo by a mutation in the asymmetric spacer region of the distal loxP site

The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region “ATAACTTCGTATA ‐NNNTANNN‐TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3′) loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl‐tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue‐specific promoters: calcium/calmodulin‐dependent kinase II alpha (Camk2a‐cre) and myosin light polypeptide 1 (Myl1‐cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt^(loxP/loxP) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695–700, 2015. © 2015 Wiley Periodicals, Inc.     

Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications

Chromosomal rearrangements are important resources for genetic studies. Recently, a Cre-loxP-based method to introduce defined chromosomal rearrangements (deletions, duplications, and inversions) into the mouse genome (chromosome engineering) has been established. To explore the limits of this technology systematically, we have evaluated this strategy on mouse chromosome 11. Although the efficiency of Cre-loxP-mediated recombination decreases with increasing genetic distance when the two endpoints are on the same chromosome, the efficiency is not limiting even when the genetic distance is maximized. Rearrangements encompassing up to three quarters of chromosome 11 have been constructed in mouse embryonic stem (ES) cells. While larger deletions may lead to ES cell lethality, smaller deletions can be produced very efficiently both in ES cells and in vivo in a tissue- or cell-type-specific manner. We conclude that any chromosomal rearrangement can be made in ES cells with the Cre-loxP strategy provided that it does not affect cell viability. In vivo chromosome engineering can be potentially used to achieve somatic losses of heterozygosity in creating mouse models of human cancers.     

Conditional RNAi in mice

RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a fast alternative to conventional knockout approaches. We developed a strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time and tissue dependent manner. Alternatively doxycycline controlled shRNA expression vectors can be used for conditional gene silencing. Single copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase mediated cassette exchange and transmitted through the germline of chimeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vectors. The site specific insertion of single copy shRNA vectors allows the expedite and reproducible production of knockdown mice and provides an easy and fast approach to assess gene function in vivo.