Germline transmission and efficient DNA recombination in mouse embryonic stem cells mediated by adenoviral-Cre transduction

Following gene targeting, a loxP-neo-loxP cassette was introduced into ES cells. The presence of a selectable marker such as neo in the targeted allele may result in gene interference in flox mice or unexpected phenotypes due to genetic ambiguity in direct knockout mice. Typically, the neo cassette is selectively removed by transient expression of the Cre recombinase in targeted ES cell. However, this method involves a tedious process of selecting, expanding, and screening ES cell clones which may compromise germline competency. Here, we describe a novel method of combining adenovirus-Cre mediated gene recombination with ES gene targeting to facilitate efficient loxP-neo-loxP removal in ES cells. We demonstrate that adenovirus-Cre infected ES cells can retain their germline competency. The procedures described here facilitate a rapid genetic manipulation of ES cells to obtain neo-free knockout animals, multiple gene targeting, homozygous mutant ES cells ideal for in vitro characterization, or Rag-deficient blastocyst complementation.     

Pooling and PCR as a method to combat low frequency gene targeting in mouse embryonic stem cells

The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.     

Heterogeneous populations of ES cells in the generation of a floxed Presenilin-1 allele

Generation of a floxed Presenilin-1 (PS1) allele involved two recombination events in the embryonic stem (ES) cells. First, a targeting vector containing a loxP site in intron 1 and a floxed CMV-HYG/TK double selection cassette in intron 3 was integrated into the PS1 locus by homologous recombination. The use of a negative selection cassette, PGK-DTA, dramatically increased the recombination efficiency within the targeted locus (75-fold). Second, an expression vector encoding Cre recombinase was introduced to excise the floxed CMV-HYG/TK cassette via site-specific recombination. However, all five ES cell clones testing positive for the proper removal of the CMV-HYG/TK cassette also contained a proportion of ES cells in which recombination had occurred between the distal loxP sites in introns 1 and 3, resulting in excision of the entire floxed region. It is therefore critical to screen for possible recombination events involving all 3 loxP sites, in order to identify ES cells clones bearing high proportions of the desired ES cells. genesis 26:5-8, 2000.     

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

The 21-23 nucleotide, single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in diverse cellular and developmental processes. In contrast to the situation for protein-coding genes, no public resource of miRNA mouse mutant alleles exists. Here we describe a collection of 428 miRNA targeting vectors covering 476 of the miRNA genes annotated in the miRBase registry. Using these vectors, we generated a library of highly germline-transmissible C57BL/6N mouse embryonic stem (ES) cell clones harboring targeted deletions for 392 miRNA genes. For most of these targeted clones, chimerism and germline transmission can be scored through a coat color marker. The targeted alleles have been designed to be adaptable research tools that can be efficiently altered by recombinase-mediated cassette exchange to create reporter, conditional and other allelic variants. This miRNA knockout (mirKO) resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community.     

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.     

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.     

介绍一种鉴定胚胎干细胞基因打靶快速、经济的筛选新方法

小鼠基因剔除动物模型越来越广泛地应用于哺乳动物基因功能与疾病的研究。然而每当胚胎干细胞同源重组的效率过低时,鉴定与分离带有定位变异的阳性克隆就会既费力又昂贵。本工作以类固醇受体共激活子基因为例,研究出一种快速鉴定阳性克隆的新方法。在构造重组载体时,将一段编码半乳糖苷酶的DNA序列整合到共激活子基因的蛋白起始码后面。于是,在干细胞内同源重组发生以后,半乳糖苷酶的表达就会受控于内源性共激活子基因的启动子。在载体与半乳糖苷酶DNA随机整合的大多数非特异克隆中,因为缺少启动子或由于不正确的氨基酸编码连接,导致合成半乳糖苷酶的可能性较小。因此,在半乳糖苷酶染色阳性的克隆中,具有特异突变的阳性克隆可以富集30倍以上。从半乳糖苷酶的阳性克隆中,再用Southern Blot方法进一步确认带有基因剔除的阳性克隆就大大减少了工作量。因为半乳糖苷酶的细胞化学染色法简便而可靠,所以在重组效率低时,可以用这种方法在短期内筛选大量克隆。但是应该注意,应用该方法的前提条件是所研究的基因必须在胚胎干细胞内表达。这些方法更为重要的意义在于当带有基因剔除的胚胎干细胞发育成小鼠后,半乳糖苷酶的组化染色法可以轻而易举地用来揭示所研究基因在动物不同组织与细胞中的表达水平。     

Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats

One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electropo-rated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targetin     

基因打靶突变小鼠与基因功能研究

ES细胞是建立基因打靶突变小鼠的必要条件 ,也可用于制备转基因动物 .基因敲除、精细突变和条件性基因打靶技术建立的基因打靶突变小鼠在人类遗传病机理研究、基因治疗和基因功能研究方面都有着重要作用 .     

Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection

Recombinase-mediated cassette exchange (RMCE), when applied to mouse embryonic stem (ES) cells, promises to increase the ease with which genetic alterations can be introduced into targeted genomic loci in the mouse. However, existing selection strategies for identifying ES cells in which replacement DNA cassettes from a carrier plasmid have been exchanged correctly into a defined locus are suboptimal. Here, we report the generation in mouse ES cells of a loxed cassette acceptor (LCA) allele within the glucokinase (gk) gene locus. Using the gkLCA as a test allele, we developed a staggered positive-negative selection strategy that facilitates efficient identification of ES cell clones in which a DNA replacement cassette from a carrier plasmid has been exchanged correctly into the gkLCA allele. This selection strategy, by facilitating more efficient production of ES cell clones with various replacement DNA cassettes, should accelerate targeted repetitive introduction of gene modifications into the mouse.     

Molecular Characterization of Mutant Mouse Strains Generated from the EUCOMM/KOMP-CSD ES Cell Resource

The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3′ LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3′ LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.     

Identification and targeting of the ROSA26 locus in human embryonic stem cells

The derivation of human embryonic stem (hES) cells has opened new avenues for studies on human development and provided a potential source of cells for replacement therapy. To reveal the full potential of hES cells, it would be advantageous to be able to genetically alter them as is routinely done with mouse ES cells through homologous recombination. The mouse Rosa26 locus is particularly useful for genetic modification as it can be targeted with high efficiency and is expressed in most cell types tested. Here we report the identification of the human homolog of the mouse Rosa26 locus. We demonstrate targeting of a red-fluorescent protein (tdRFP) cDNA to this locus through homologous recombination and expression of this targeted reporter in multiple hES cell-derived lineages. Through recombinase-mediated cassette exchange, we show replacement of the tdRFP cDNA with other cDNAs, providing a cell line in which transgenes can be readily introduced into a broadly expressed locus.     

TECHNICAL REPORT: Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques

Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells     

Efficient gene targeting by homologous recombination in rat embryonic stem cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat.