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Turan S Galla M Ernst E Qiao J Voelkel C Schiedlmeier B Zehe C Bode J 《Journal of molecular biology》2011,407(2):193-17240
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. 相似文献
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Sathiya N. Manivannan Thomas L. Jacobsen Peter Lyon Bhavani Selvaraj Peter Halpin Amanda Simcox 《Genetics》2015,201(4):1319-1328
Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied. 相似文献
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Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology
Soeren Turan 《Journal of molecular biology》2010,402(1):52-69
There are strong indications, but as yet no proof, that extended 48-bp Flp recombinase targets (FRTs) represent unique targets in all eukaryotic genomes investigated, and that recombinase-mediated cassette exchange is not hampered by the occurrence of genomic pseudo sites. This encouraged the present study in which we explore the feasibility of exchanging, in a given cell, two distinct genomically anchored cassettes, each flanked by a unique set of two heterospecific FRT sites. Mutant FRTs have to meet two major prerequisites for successful recombinase-mediated cassette exchange: (i) a self-recognition capacity comparable to a pair of FRT wild-type sites (FRT × FRT ), and (ii) a negligible cross-interaction if part of a set of heterospecific sites (F′ × F). We apply a two-step strategy to explore various newly created FRT spacer mutants for these properties. As a result of our screening steps, we identify combinations of sites that are successfully applied to parallel Flp-mediated genomic targeting (“multiplexing”) reactions (i.e., the simultaneous exchange of two separate target cassettes in a given cell). 相似文献
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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. 相似文献
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Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is “clean” in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (∼ 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype. 相似文献
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Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection 总被引:3,自引:0,他引:3
Long Q Shelton KD Lindner J Jones JR Magnuson MA 《Genesis (New York, N.Y. : 2000)》2004,39(4):256-262
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. 相似文献
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Masato Ohtsuka Hiromi Miura Keiji Mochida Michiko Hirose Ayumi Hasegawa Atsuo Ogura Ryuta Mizutani Minoru Kimura Ayako Isotani Masahito Ikawa Masahiro Sato Channabasavaiah B Gurumurthy 《BMC genomics》2015,16(1)
Background
The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.Results
Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.Conclusions
The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users. 相似文献9.
Zhongsen Li Aiqiu Xing Bryan P. Moon Richard P. McCardell Kelly Mills S. Carl Falco 《Plant physiology》2009,151(3):1087-1095
A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.Plant transformation has challenges such as random integration, multiple transgene copies, and unpredictable expression. Homologous recombination (Iida and Terada, 2005; Wright et al., 2005) and DNA recombinase-mediated site-specific integration (SSI) are promising technologies to address the challenges for placing a single copy of transgenes into a precharacterized site in a plant genome.Several site-specific DNA recombination systems, such as the bacteriophage Cre-lox and the yeast FLP-FRT and R-RS, have been used in SSI studies (Ow, 2002; Groth and Calos, 2003). A common feature of these systems is that each system consists of a recombinase Cre, FLP, or R and two identical or similar palindromic recognition sites, lox, FRT, or RS. Each recognition site contains a short asymmetric spacer sequence where DNA strand exchange takes place, flanked by inverted repeat sequences where the corresponding recombinase specifically binds. If two recognition sites are located in cis on a DNA molecule, the DNA segment can be excised if flanked by two directionally oriented sites or inverted if flanked by two oppositely oriented sites. If two recognition sites are located in trans on two different DNA molecules, a reciprocal translocation can happen between the two DNA molecules or the two molecules can integrate if at least one of them is a circular DNA (Ow, 2002; Groth and Calos, 2003).Single-site SSI can integrate a circular donor DNA containing one recognition site into a similar site previously placed in a plant genome. The integrated transgene now flanked by two recognition sites is vulnerable to excision. Transient Cre expression and the use of mutant lox sites to create two less compatible sites after integration helped reduce the subsequent excision in tobacco (Nicotiana tabacum; Albert et al., 1995; Day et al., 2000). A similar approach was used to produce SSI events in rice (Oryza sativa), and the transgene was proven stably expressed over generations (Srivastava and Ow, 2001; Srivastava et al., 2004; Chawla et al., 2006). Using a promoter trap to displace a cre gene in the genome with a selection gene from the donor, approximately 2% SSI was achieved in Arabidopsis (Arabidopsis thaliana; Vergunst et al., 1998).When two recognition sites located on a linear DNA molecule are similar enough to be recognized by the same recombinase but different enough to reduce or prevent DNA recombination from happening between them, the DNA segment between the two sites may not be easily excised or inverted. When a circular DNA molecule carrying the same two incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000; Baer and Bode, 2001).RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al., 2005). RMCE using both the Cre-lox and FLP-FRT systems improved RMCE frequency in animal cell cultures (Lauth et al., 2002). RMCE using two directly oriented incompatible FRT sites and transiently expressed FLP recombinase achieved cassette exchange between a target previously placed in the Drosophila genome and a donor introduced as a circular DNA (Horn and Handler, 2005). A gene conversion approach involving Cre-lox- and FLP-FRT-mediated SSI, RMCE, and homologous recombination was explored in maize (Zea mays; Djukanovic et al., 2006). RMCE using two oppositely oriented incompatible lox sites and transiently expressed Cre recombinase produced single-copy RMCE plants in Arabidopsis (Louwerse et al., 2007).To develop FLP-FRT-mediated RMCE in soybean (Glycine max), we created transgenic target lines containing a hygromycin resistance gene flanked by two directly oriented incompatible FRT sites via biolistic transformation. Single-copy target lines were selected and retransformed with a donor DNA containing a chlorsulfuron resistance gene flanked by the same pair of FRT sites. An FLP expression DNA was cobombarded to transiently provide FLP recombinase. RMCE events were obtained from multiple target lines and confirmed by extensive molecular characterization. 相似文献
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Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology 总被引:1,自引:0,他引:1
André Oumard Junhua Qiao Thomas Jostock Jiandong Li Juergen Bode 《Cytotechnology》2006,50(1-3):93-108
The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like ΦC31, which recombine attP and attB target sites in a one-way reaction – at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and ΦC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is ‘clean’ in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker. 相似文献
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Shuohao Huang Yoshinori Kawabe Akira Ito Masamichi Kamihira 《Biotechnology and bioengineering》2010,107(4):717-729
Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase‐mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild‐type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase‐defective retroviral vectors (IDRV), which contains an ATG‐deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418‐resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site‐specific recombination. The successful substitution of the original viral integration machinery with a non‐viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717–729. © 2010 Wiley Periodicals, Inc. 相似文献
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Matthew T. O’Neill Julie Healer Alan F. Cowman 《International journal for parasitology》2011,41(1):117-123
The ability to manipulate the genome and induce site-specific recombination using either Flippase (FLP) or Cre recombinase has been useful in many systems including Plasmodium berghei for specific deletion events or to obtain conditional gene expression. To test whether these recombinases are active in Plasmodium falciparum we constructed gene knockouts that contain sequences recognised as templates for site-specific recombination. We tested the ability of FLP and Cre recombinases, expressed conditionally in P. falciparum, to mediate deletion of the human dihydrofolate reductase (hdhfr) drug resistance gene. We show that Cre recombinase is capable of efficient removal of hdhfr by site-specific recombination. In contrast, FLP recombinase is very inefficient, even at the optimum temperature of 30 °C for this enzyme. These results demonstrate that Cre recombinase can be utilised in P. falciparum for deletion of specific sequences such as drug resistance genes. This can be exploited for recycling of drug resistance cassettes and for the design of specific recombination events in P. falciparum. 相似文献
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Monsur Alam Khan Mohammad Monirul Islam Yutaka Kuroda 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2107-2115
Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein. 相似文献
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The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator. 相似文献
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Animal genomes contain ∼20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev, and Transib. 相似文献
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Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites 总被引:2,自引:0,他引:2
Bolusani S Ma CH Paek A Konieczka JH Jayaram M Voziyanov Y 《Nucleic acids research》2006,34(18):5259-5269
As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites. 相似文献
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Total genomic DNA from Bursaphelenchus xylophilus pathotypes MPSy-1 and VPSt-1 and from B. mucronatus was digested with restriction endonucleases. DNA fragments were electrophoretically separated, Southern blotted to nitrocellulose, and hybridized to genomic DNA from one of the isolates. The resulting hybridization patterns indicate genomic differences in repetitive DNA sequences among these populations. Greatest differences were seen between B. xylophilus and B. mucronatus, but genomic differences were also apparent between B. xylophilus pathotypes MPSy-1 and VPSt-1 and between a population from P. nigra in New Jersey and a population of a mucronate form from Abies balsamea in Quebec, Canada. 相似文献
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Gilberto Vargas-Alarcon Maite Vallejo Carlos Posadas-Romero Juan G. Juarez-Rojas Marco Antonio Martinez-Rios Marco Antonio Peña-Duque Silvia Carrillo-Sanchez Oscar Perez-Mendez José Manuel Fragoso 《Gene》2014
Endothelial dysfunction plays an essential role in the development and progression of atherosclerotic lesions. Endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) are considered important molecules in the endothelial dysfunction process. The aim of the present study was to evaluate the role of eNOS and ET-1 (EDN1) gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS). Six polymorphisms (rs1799983, rs2070744, rs1800783, rs3087459, rs1800541, and rs5369) of eNOS and EDN1 genes were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 452 patients with ACS and 283 healthy controls. The results showed increased frequencies of the A allele of the END1-914 C>A (rs3087459) polymorphism in ACS patients when compared to controls (OR = 1.56, Pc = 0.01). Under an additive model, the “AA” genotype was associated with an increased risk of developing ACS, adjusted for gender, hypertension, dyslipidemia, alcohol consumption, smoking, and diabetes (OR = 1.56, p = 0.045). Linkage disequilibrium analysis showed one EDN1 haplotype (AT) with increased frequency in ACS patients when compared to healthy controls (OR = 1.65, Pc = 0.0015). The “AT” haplotype was associated with the risk of developing ACS after adjusting for cardiovascular risk factors using multiple logistic analysis. In this case, the adjusted OR was 1.73 for the AT haplotype (Pc = 0.0018). In summary, resulting data suggest that the END1-914 C>A gene polymorphism could be involved in the risk of developing ACS in Mexican individuals. 相似文献