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.     

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.     

Conditional transgene expression mediated by the mouse beta-actin locus

Transgenic mice are an effective model to study gene function in vivo; however, position effects can complicate tissue-specific transgene analysis. To facilitate precise targeting of a transgenic construct into the mouse genome, we combined the Cre/lox and Flp/FRT recombination systems to allow for rapid transgene replacement and conditional transgene expression from the endogenous beta-actin locus. Flp/FRT recombination was used to rapidly exchange FRT-flanked transgene cassettes by recombinase-mediated cassette exchange in embryonic stem cells, while transgene expression can be activated in mice after Cre-mediated excision of a floxed STOP cassette. To validate our system, we analyzed the expression profile of an EGFP reporter gene after integration into the beta-actin locus and Cre-mediated excision of the floxed STOP cassette. Breeding of EGFP reporter mice with various Cre mouse lines resulted in the expected expression profiles, demonstrating the feasibility of the model to facilitate predictable and strong transgene expression in a spatially and temporally controlled manner.     

Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development

Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY-->AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR-->AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL-->AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.     

Sequential genetic modification of the hprt locus in human ESCs combining gene targeting and recombinase-mediated cassette exchange

Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line.     

Targeted engineering of the Drosophila genome

The application of phiC31 phage integrase in Drosophila for unidirectional and site-specific DNA integration was pioneered by Groth et al. in 2004 1 and quickly triggered a wave of innovative tools taking advantage of these unique properties of phiC31. Three recent papers have further developed novel approaches that combine the phiC31-mediated DNA integration with the homologous recombination (HR)-based gene targeting 2 3 for the purpose of efficient and targeted modifications of Drosophila genomic loci. Despite significant differences, the general strategies are similar in principle in the SIRT (site-specific integrase mediated repeated targeting) approach by Gao et al. 4, the IMAGO (integrase-mediated approach for gene knock-out) approach by Choi et al. 5 and the genomic engineering approach developed by our group 6. All three use HR-based gene targeting to first implant a single or a pair of phiC31-attP recombination sites into the target locus. Flies carrying such targeted insertions of attP sites can then be used as "founder lines", in which modified DNA sequences ("knock-in DNA") can be repeatedly and efficiently inserted back into the target locus via phiC31-mediated integration. Thus, by carrying out the targeting experiments only once, one can then directedly and efficiently modify the target locus into virtually any desired knock-in allele. Here we give a brief overview of the SIRT, IMAGO, and genomic engineering approaches and propose a revised genomic engineering scheme in which a single ends-out targeting event will generate founder lines suitable for both recombinase-mediated cassette exchange (RMCE) and single-site based integration of knock-in DNA.     

Transposon-mediated BAC transgenesis in zebrafish

Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ～15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.     

基于Flp重组酶介导的盒式交换HPRT位点定点转基因研究

Exchange plasmid pF-HPRT-F3 and Flp expression plasmid pCMV-Flp were constructed and then introduced using electroporation system into F18 ES cell line, which have an exchange cassette F-Neo-F3 at HPRT locus. After HAT selection, HAT resistant clones were obtained. Then G418 sensitivity test and Southern blotting were carried out to screen RMCE recombinants. The results indicated that RMCE had taken place in three of 12 HAT resistant clones. The frequency is 25%. The result demonstrates that it is realizable to introduce transgene to HPRT locus by using Flp recombinase mediated cassette exchange reaction.     

基于Flp重组酶介导的盒式交换HPRT位点定点转基因研究

以电穿孔转染法将构建的交换质粒pF-HPRT-F3和Flp表达载体pCMV-Flp共转染已在基因组HPRT位点整合有交换盒F-Neo-F3结构的ES细胞F18,经HAT筛选得到HAT抗性ES细胞克隆;G418敏感性试验和基因组Southern杂交表明,所分析的12个HAT抗性克隆中有3个克隆发生了预期的盒式交换重组,转基因定点整合的相对效率为25%.这一结果表明,Flp重组酶是可以在HPRT位点有效地介导盒式交换反应的,我们提出的利用Flp重组酶介导的盒式交换反应建立基于HPRT位点的转基因定点整合体系的策略是可行的.     

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.     

Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.     

Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system

We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor plasmids including an expression cassette for an antibody gene flanked by a compatible pair of loxP sites were prepared. The donor plasmid and a Cre expression vector were co-transfected into the founder CHO cells to give rise to RMCE in the CHO genome, resulting in site-specific integration of the antibody gene. The RMCE procedure was repeated to increase the copy numbers of the integrated gene. Southern blot and genomic PCR analyses for the established cells revealed that the transgenes were integrated into the target site. Antibody production determined by ELISA and western blotting was increased corresponding to the number of transgenes. These results indicate that the accumulative site-specific gene integration system could provide a useful tool for increasing the productivity of recombinant proteins.     

Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges

Traditional DNA transduction routes used for the modification of cellular genomes are subject to unpredictable alterations, as the cell-intrinsic repair machinery may affect both the integrity of the transgene and the recipient locus. These problems are overcome by recombinase-mediated cassette exchange (RMCE) approaches enabling predictable expression patterns by the nondisruptive insertion of a gene cassette at a pre-characterized genomic locus. The destination is marked by a “tag” consisting of two heterospecific recombination target sites (RTs) at the flanks of a selection marker. Provided on a circular donor vector, an analogous cassette encoding the gene of interest can cleanly replace the resident cassette under the influence of a site-specific recombinase. RMCE was first based on the yeast integrase Flp but had to give way to the originally more active phage-derived Cre enzyme. To be effective, both Tyr-recombinases have to be applied at a considerable concentration, which, in the case of Cre, triggers endonucleolytic activities and therefore cellular toxicity. This review addresses the particularities of both recombination routes depending on the structure of the synaptic complex and on improved integrase and RT variants. While the performance of Flp-RMCE can now firmly rely on optimized Flp variants and multiple sets of functional target sites (FRTs), the Cre system suffers from the promiscuity of its RT mutants, which is explained in molecular terms. At present, RMCE enters applications in the stem cell field. Remarkable efforts are noted in the framework of various mouse mutagenesis programs, which, in their first phase, have targeted virtually all genes and now start to shift their emphasis from gene trapping to gene modification.     

A simple polymerase chain reaction-based method for the construction of recombinase-mediated cassette exchange donor vectors

Here we describe a simple method for generating donor vectors suitable for targeted transgenesis via recombinase-mediated cassette exchange (RMCE) using the ΦC31 integrase. This PCR-based strategy employs small attB “tails” on the primers used to amplify a sequence of interest, permitting the rapid creation of transgenes for in vivo analysis.     

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.     

Efficient Flp-Int HK022 dual RMCE in mammalian cells

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.     

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.