363 results on '"Site-specific recombinase technology"'
Search Results
2. Chemical Controllable Gene Drive in Drosophila
- Author
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Seok Jun Moon, Nayoung Lee, Dongwoo Chae, Hyongbum Kim, Junwon Lee, and Kyungsoo Park
- Subjects
0106 biological sciences ,0303 health sciences ,Transgene ,fungi ,Biomedical Engineering ,Inheritance (genetic algorithm) ,food and beverages ,General Medicine ,Computational biology ,Gene drive ,Biology ,biology.organism_classification ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,03 medical and health sciences ,010608 biotechnology ,Recombinase ,CRISPR ,Site-specific recombinase technology ,Gene ,Drosophila ,030304 developmental biology - Abstract
Gene drive systems that propagate transgenes via super-Mendelian inheritance can potentially control insect-borne diseases and agricultural pests. However, concerns have been raised regarding unforeseen ecological consequences, and methods that prevent undesirable gene drive effects have been proposed. Here, we report a chemical-induced control of gene drive. We prepared a CRISPR-based gene drive system that can be removed by a site-specific recombinase, Rippase, the expression of which is induced by the chemical RU486 in fruit flies. Exposure of fruit flies to RU486 resulted in 7-12% removal of gene drive elements at each generation, leading to a significant reduction in gene drive-fly propagation. Mathematical modeling and simulation suggest that our system offers several advantages over a previously reported gene drive control system. Our chemical control system can provide a proof-of-principle for the reversible control of gene drive effects depending on ecological status and human needs.
- Published
- 2020
3. Production of marker-free cisgenic apple plants using inducible site-specific recombinase and a bifunctional selectable gene
- Author
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S.V. Dolgov, V.R. Timerbaev, and T.Y. Mitiouchkina
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chemistry.chemical_compound ,chemistry ,Biochemistry ,Cisgenesis ,Site-specific recombinase technology ,Horticulture ,Biology ,Marker free ,Bifunctional ,Gene - Published
- 2019
4. Short-Term and Highly Efficient Cas9 Expression System Using Adenovirus Vector and Site-Specific Recombinase Cre
- Author
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Kazunori Akimoto, Yumi Kanegae, Emi Tsuchitani, Saki Kondo, Sayaka Nagamoto, and Miyuki Agawa
- Subjects
Cas9 ,Site-specific recombinase technology ,Biology ,Term (time) ,Viral vector ,Cell biology - Abstract
Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least six months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80–90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.
- Published
- 2021
5. Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution
- Author
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Xueying Tian and Bin Zhou
- Subjects
0301 basic medicine ,ER, estrogen receptor ,Light ,Computer science ,CRY2, cryptochrome 2 ,Biochemistry ,Genetic recombination ,Regenerative medicine ,BphP1, bacterial phytochrome ,PA-Cre, photoactivatable Cre recombinase ,Recombinase ,Dox, doxycycline ,Promoter Regions, Genetic ,site-specific recombinase ,Recombination, Genetic ,site-specific recombination ,CreC, the C-terminal domain of Cre ,CreN, the N-terminal domain of Cre ,CrexER, a switchable CreER system with the Cre-rox-ER-rox construct ,FISC system, far-red light-induced split Cre-loxP system ,dRap, light-cleavable rapamycin dimer ,Plants ,sCreER, self-cleaved inducible CreER ,iSuRe-Cre, Cre/CreERT2-inducible dual Reporter-Cre-expressing mouse allele ,Roxed-Cre, a sequential binary SSR system with the Cre-N-rox-stop-rox-Cre-C strategy ,SSR, site-specific recombinase ,DNA Nucleotidyltransferases ,Dre-rox ,TRE, Tet responsive element ,PIF, photochrome-interacting factor ,Tet-On system, tetracycline-inducible gene expression system ,DHT, dihydrotestosterone ,PhyB, photoreceptor phytochrome B ,rtTA, reverse tet-controlled transactivator ,4-OHT, 4-hydroxytamoxifen ,LBD, ligand binding domain ,Computational biology ,Li-rtTA, light activated rtTA ,Catalysis ,Genome engineering ,nMag, negative magnet ,03 medical and health sciences ,lineage tracing ,PpsR2, the natural partner of BphP1 ,inducible ,Hsp, heat shock protein ,Site-specific recombinase technology ,Site-specific recombination ,VVD, photoreceptor Vivid ,optogenetics ,Molecular Biology ,photoactivatable ,gene manipulation ,GR, glucocorticoid receptor ,030102 biochemistry & molecular biology ,LightOn, the light-on system ,Integrases ,JBC Reviews ,PR, progesterone receptor ,Cell Biology ,Di-Cre, dimerizable Cre ,Enzyme Activation ,pMag, positive magnet ,030104 developmental biology ,Cre-loxP ,AR, androgen receptor ,Cre-Lox recombination ,HR, homologous recombination ,CRISPR-Cas Systems ,Homologous recombination ,genome engineering ,CIB1, a basic helix-loop-helix protein - Abstract
Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.
- Published
- 2021
6. Efficient Gene Stacking in Rice Using the GAANTRY System
- Author
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James G. Thomson, Roger Thilmony, James Horstman, and Leyla T. Hathwaik
- Subjects
Gene stacking ,Oryza sativa ,Site-specific recombinase ,biology ,Agrobacterium ,Transgene ,Soil Science ,food and beverages ,Plant Science ,Computational biology ,lcsh:Plant culture ,biology.organism_classification ,Plasmid ,Arabidopsis ,Genetic engineering ,Recombinase ,Site-specific recombinase technology ,lcsh:SB1-1110 ,Original Article ,Agronomy and Crop Science ,Gene - Abstract
Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.
- Published
- 2020
7. Beyond knockouts: Applying precision genome editing for conditional mutagenesis innovation in zebrafish
- Author
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Maira Pedroso de Almeida
- Subjects
GAL4/UAS system ,Genome editing ,Spatiotemporal Analysis ,CRISPR ,Site-specific recombinase technology ,Computational biology ,Biology ,biology.organism_classification ,Conditional mutagenesis ,Zebrafish ,Gene knockout - Published
- 2020
8. Site‐specific recombinase genome engineering toolkit in maize
- Author
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Jon P. Cody, Changzeng Zhao, Nathan C. Swyers, Nathaniel D. Graham, and James A. Birchler
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0106 biological sciences ,Transgene ,Agrobacterium ,Plant Science ,Computational biology ,Biology ,bombardment ,maize ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Genome engineering ,03 medical and health sciences ,Recombinase ,Site-specific recombinase technology ,Gene ,Ecology, Evolution, Behavior and Systematics ,Selectable marker ,Original Research ,030304 developmental biology ,2. Zero hunger ,genetic engineering ,0303 health sciences ,Ecology ,recombinases ,fungi ,Botany ,Integrase ,QK1-989 ,biology.protein ,Excisionase ,010606 plant biology & botany - Abstract
Site‐specific recombinase enzymes function in heterologous cellular environments to initiate strand‐switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium‐mediated transformation of T‐DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking.
- Published
- 2020
9. Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440
- Author
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Gara N. Wolff, Anna Furches, Kent Gorday, Adam M. Guss, and Joshua R. Elmore
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0301 basic medicine ,Endocrinology, Diabetes and Metabolism ,lcsh:Biotechnology ,Biomedical Engineering ,Biology ,Promoter library ,Genome ,Article ,03 medical and health sciences ,0302 clinical medicine ,Plasmid ,lcsh:TP248.13-248.65 ,Recombinase ,Site-specific recombinase technology ,Gene ,lcsh:QH301-705.5 ,Genetics ,Site-specific recombinase ,Pseudomonas putida ,biology.organism_classification ,030104 developmental biology ,lcsh:Biology (General) ,Genetic engineering ,Heterologous expression ,Gene expression ,Homologous recombination ,030217 neurology & neurosurgery - Abstract
Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Further, most heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. To improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, −35 sequences, and −10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. An additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering., Highlights • BxB1 integrase catalyzes site-specific DNA integration into P. putida chromosome. • Promoter library (−35/−10 variants) covers a 72-fold range of protein expression. • Expression can be further tuned by 2-fold in P. putida with RBS and UP-elements.
- Published
- 2017
10. The Gene or Not the Gene—That Is the Question: Understanding the Genetically Engineered Mouse Phenotype.
- Author
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Sellers, R. S.
- Subjects
EMBRYONIC stem cells ,PHENOTYPES ,LABORATORY mice ,TRANSGENIC mice ,GENETICS - Abstract
This article discusses embryonic stem (ES) cells. The use and knowledge of the ES cells nowadays are mentioned as coming from earlier works on cancer cells involving mice by, among others, Leroy Stevens at the Jackson Labs and Kleinsmith and Pierce. The early beginning of transgenic technology in the early 1970s is related. The article also addresses the partial replacement of ES cell technology by zinc finger nuclease technology and looks as well into the future of mutant mouse.
- Published
- 2012
- Full Text
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11. Transgene stacking in potato using the GAANTRY system
- Author
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James G. Thomson, Ethan Gardner, Roger Thilmony, Kent F. McCue, and Ronald Chan
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DNA, Bacterial ,0301 basic medicine ,Agrobacterium ,Transgene ,Gene Dosage ,Gene Expression ,lcsh:Medicine ,Genetically modified crops ,Polymerase Chain Reaction ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Plasmid ,Genes, Reporter ,Recombinase ,Site-specific recombinase technology ,Transgenes ,030212 general & internal medicine ,GAANTRY ,lcsh:Science (General) ,lcsh:QH301-705.5 ,Glucuronidase ,Gene-stacking ,Solanum tuberosum ,Site-specific recombinase ,biology ,lcsh:R ,fungi ,Gene Transfer Techniques ,food and beverages ,General Medicine ,Plants, Genetically Modified ,biology.organism_classification ,Cell biology ,Plant Leaves ,Research Note ,Luminescent Proteins ,Transformation (genetics) ,Phenotype ,030104 developmental biology ,lcsh:Biology (General) ,Plasmids ,lcsh:Q1-390 ,Transformation efficiency - Abstract
Objective GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA). We examined the ability of the GAANTRY Agrobacterium rhizogenes ArPORT1 ‘10-stack’ strain to generate transgenic potato plants. Results The 28.5 kilobase 10-stack T-DNA, was introduced into Lenape potato plants with a 32% transformation efficiency. Molecular and phenotypic characterization confirmed that six of the seven tested independent transgenic lines carried the entire desired construct, demonstrating that the GAANTRY 10-stack strain can be used can be used in a tissue culture-based callus transformation method to efficiently generate transgenic potato plants. Analysis using droplet digital PCR showed that most of the characterized events carry one or two copies of the 10-stack transgenes and that ‘backbone’ DNA from outside of the T-DNA was absent in the transgenic plants. These results demonstrate that the GAANTRY system efficiently generates high quality transgenic potato plants with a large construct of stacked transgenes. Electronic supplementary material The online version of this article (10.1186/s13104-019-4493-8) contains supplementary material, which is available to authorized users.
- Published
- 2019
12. Efficient generation of selection-gene-free rat knockout models by homologous recombination in ES cells
- Author
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Elizabeth C. Bryda, Yuanyuan Wu, Shuping Li, He Lan, Ning Li, Hongsheng Men, Mario R. Capecchi, Zihang Guo, and Sen Wu
- Subjects
Leptin ,Male ,0301 basic medicine ,Knockout rat ,Regulation of Gene Expression ,Cell ,Biophysics ,homologous recombination ,Biology ,Biochemistry ,gene targeting ,Cell Line ,Rats, Sprague-Dawley ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Research Letter ,Genetics ,medicine ,Animals ,rat ,Site-specific recombinase technology ,Molecular Biology ,Gene ,Embryonic Stem Cells ,Gene targeting ,selection‐gene‐free ,Cell Biology ,ES cells ,Molecular biology ,Embryonic stem cell ,Research Letters ,Rats, Inbred F344 ,Rats ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Cell culture ,Tumor Suppressor Protein p53 ,Homologous recombination ,030217 neurology & neurosurgery - Abstract
Embryonic stem cell (ES cell)-based rat knockout technology, although successfully developed in 2010, has seen very limited usage to date due to low targeting efficiency and a lack of optimized procedures. In this study, we performed gene targeting in ES cells from the Sprague-Dawley (SD) and the Fischer 344 (F344) rat strains using an optimized procedure and the self-excising neomycin (neo)-positive selection cassette ACN to successfully generate Leptin and Trp53 knockout rats that did not carry the selection gene. These results demonstrate that our simplified targeting strategy using ACN provides an efficient approach to knock out many other rat genes.
- Published
- 2016
13. Mechanisms of gene targeting in higher eukaryotes
- Author
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Hirofumi Anai, Katsuhiro Hanada, and Akinori Tokunaga
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0301 basic medicine ,Pharmacology ,Genetics ,Deoxyribonucleases ,Models, Genetic ,Eukaryota ,Gene targeting ,Cell Biology ,Protein engineering ,Biology ,Protein Engineering ,Genome ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,030104 developmental biology ,Genome editing ,Adherent cell ,Gene Targeting ,Molecular Medicine ,DNA Breaks, Double-Stranded ,Site-specific recombinase technology ,Genomic information ,Homologous Recombination ,Homologous recombination ,Molecular Biology - Abstract
Targeted genome modifications using techniques that alter the genomic information of interest have contributed to multiple studies in both basic and applied biology. Traditionally, in gene targeting, the target-site integration of a targeting vector by homologous recombination is used. However, this strategy has several technical problems. The first problem is the extremely low frequency of gene targeting, which makes obtaining recombinant clones an extremely labor intensive task. The second issue is the limited number of biomaterials to which gene targeting can be applied. Traditional gene targeting hardly occurs in most of the human adherent cell lines. However, a new approach using designer nucleases that can introduce site-specific double-strand breaks in genomic DNAs has increased the efficiency of gene targeting. This new method has also expanded the number of biomaterials to which gene targeting could be applied. Here, we summarize various strategies for target gene modification, including a comparison of traditional gene targeting with designer nucleases.
- Published
- 2015
14. An approach for controlling the timing and order of engineered mutations in mice
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Maxwell M. Goodrich, David W. Goodrich, Ramzi Talhouk, and Xiaojing Zhang
- Subjects
0301 basic medicine ,Cell type ,Transgene ,Cre recombinase ,Mice, Transgenic ,Computational biology ,Biology ,medicine.disease_cause ,Article ,03 medical and health sciences ,Mice ,Endocrinology ,Genetics ,medicine ,Animals ,Estrogen Receptor beta ,Site-specific recombinase technology ,Transgenes ,Gene ,Recombination, Genetic ,Mutation ,Cell Biology ,Phenotype ,Fusion protein ,Tamoxifen ,030104 developmental biology ,DNA Nucleotidyltransferases ,Mutagenesis, Site-Directed ,Genetic Engineering - Abstract
Significant advances in our understanding of normal development and disease have been facilitated by engineered mice in which genes can be altered in a spatially, temporally, or cell type restricted manner using site specific recombinase systems like Cre-loxP or Flp-frt. In many circumstances it is important to understand how interactions between multiple genes influence a given phenotype. Robust approaches for precisely controlling multiple genetic alterations independently are limited, however, thus the impact of mutation order and timing on phenotype is generally unknown. Here we describe and validate a novel Gt(ROSA)26Sor targeted transgene allowing precise control over the order and timing of multiple genetic mutations in the mouse. The transgene expresses an optimized, Flp-estrogen receptor fusion protein (Flpo-ERT2) under the control of a loxP-stop-loxP cassette. In this system, genes modified by loxP sites are altered first upon expression of Cre. Cre also eliminates the loxP-stop-loxP cassette, permitting wide-spread expression of Flpo-ERT2. Because of the estrogen receptor fusion, Flp activity remains inert until administration of tamoxifen, allowing genes modified by frt sites to be modified subsequently with controllable timing. This mouse transgene will be useful in a wide variety of applications where independent control of different mutations in the mouse is desirable.
- Published
- 2018
15. Perinatal induction of Cre recombination with tamoxifen
- Author
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Benoit Lizen, Melissa Claus, Lucie Jeannotte, Françoise Gofflot, and Filippo M. Rijli
- Subjects
Male ,medicine.drug_class ,Transgene ,Mice, Transgenic ,Context (language use) ,Biology ,Andrology ,Mice ,Pregnancy ,Genetics ,medicine ,Recombinase ,Animals ,Site-specific recombinase technology ,Homeodomain Proteins ,Recombination, Genetic ,Integrases ,Estrogen Antagonists ,Phosphoproteins ,medicine.disease ,Mice, Inbred C57BL ,Perinatal Care ,Tamoxifen ,Animals, Newborn ,Receptors, Estrogen ,Estrogen ,Immunology ,Mice, Inbred CBA ,Gestation ,Female ,Animal Science and Zoology ,Agronomy and Crop Science ,Transcription Factors ,Biotechnology ,medicine.drug - Abstract
Temporal control of site-specific recombination is commonly achieved by using a tamoxifen-inducible form of Cre or Flp recombinases. Although powerful protocols of induction have been developed for gene inactivation at adult stages or during embryonic development, induction of recombination at late gestational or early postnatal stages is still difficult to achieve. In this context, using the ubiquitous CMV-CreER(T2) transgenic mice, we have tested and validated two procedures to achieve recombination just before and just after birth. The efficiency of recombination was evaluated in the brain, which is known to be more problematic to target. For the late gestation treatment with tamoxifen, different protocols of complementary administration of progesterone and estrogen were tested. However, delayed delivery and/or mortality of pups due to difficult delivery were always observed. To circumvent this problem, pups were collected from tamoxifen-treated pregnant dams by caesarian section at E18.5 and given to foster mothers. For postnatal treatment, different dosages of tamoxifen were administered by intragastric injection to the pups during 3 or 4 days after birth. The efficiency of these treatments was analyzed at P7 using a transgenic reporter line. They were also validated with the Hoxa5 conditional allele. In conclusion, we have developed efficient procedures that allow achieving efficient recombination of floxed alleles at perinatal stages. These protocols will allow investigating the late/adult functions of many developmental genes, whose characterization has been so far restricted to embryonic development.
- Published
- 2015
16. The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis
- Author
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David G. Kirsch and Minsi Zhang
- Subjects
Male ,FLP-FRT recombination ,Medicine (miscellaneous) ,lcsh:Medicine ,Mouse models ,Genome ,Mice ,0302 clinical medicine ,Immunology and Microbiology (miscellaneous) ,Recombinase ,Homeostasis ,Genetics ,0303 health sciences ,3. Good health ,Cell biology ,030220 oncology & carcinogenesis ,DNA Nucleotidyltransferases ,Female ,Genetic Engineering ,lcsh:RB1-214 ,Cell type ,Transgene ,Sequential mutagenesis ,Neuroscience (miscellaneous) ,Mice, Transgenic ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Collagen Type I ,Adenoviridae ,03 medical and health sciences ,lcsh:Pathology ,Animals ,Site-specific recombinase technology ,Gene ,Alleles ,Embryonic Stem Cells ,030304 developmental biology ,Integrases ,lcsh:R ,Retraction ,Collagen Type I, alpha 1 Chain ,Mice, Inbred C57BL ,Disease Models, Animal ,Tamoxifen ,Microscopy, Fluorescence ,Mutagenesis ,Genetically Engineered Mouse ,Mutation ,Dual recombinase technology ,Gene Deletion - Abstract
Novel genetically engineered mouse models using the Cre-loxP or the Flp-FRT systems have generated useful reagents to manipulate the mouse genome in a temporally-regulated and tissue specific manner. By incorporating a constitutive Cre driver line into a mouse model in which FRT-regulated genes in other cells types are regulated by Flp-FRT recombinase, gene expression can be manipulated simultaneously in separate tissue compartments. This application of dual recombinase technology can be used to dissect the role of stromal cells in tumor development and cancer therapy. Generating mice in which Cre-ERT2 is expressed under Flp-FRT-mediated regulation would enable step-wise manipulation of the mouse genome using dual recombinase technology. Such next-generation mouse models would enable sequential mutagenesis to better model cancer and define genes required for tumor maintenance. Here, we generated novel genetically engineered mice that activate or delete Cre-ERT2 in response to Flp recombinase. To potentially utilize the large number of Cre-loxP regulated transgenic alleles that have already been targeted into the Rosa26 locus, such as different reporters and mutant genes, we targeted the two novel Cre-ERT2 alleles into the endogenous Col1a1 locus for ubiquitous expression. In the Col1a1FRT-Cre-ER-T2-FRT mice, Flp deletes Cre-ERT2, so that Cre-ERT2 is only expressed in cells which have never expressed Flp. In contrast, in the Col1a1FRT-STOP-FRT-Cre-ER-T2 mice, Flp removes the STOP cassette to allow Cre-ERT2 expression so that Cre-ERT2 is only expressed in cells that previously expressed Flp. These two new novel mouse strains will be complementary to each other and will enable the exploration of complex biological questions in development, normal tissue homeostasis, and cancer.
- Published
- 2015
17. TRANSFORMATION EFFECTIVENESS FOR ARABIDOPSIS THALIANA PLANTS BY DNA-CONSTRUCTIONS WITH SITE-SPECIFIC RECOMBINASE SYSTEM Cre/loxP
- Author
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A. S. Sekan and S. V. Isaenkov
- Subjects
lcsh:Biotechnology ,site-specific recombinase system Cre/loxP ,Biology ,biology.organism_classification ,Cell biology ,Transformation (genetics) ,chemistry.chemical_compound ,chemistry ,lcsh:TP248.13-248.65 ,Arabidopsis thaliana ,Site-specific recombinase technology ,Cre-Lox recombination ,marker genes excision ,DNA ,Floxing - Abstract
Using of new approach with site-specific recombinase system Cre/loxP under the control of 35S-promoter to generate marker-free genetically modified plants was developed. The analysis of recombinase system was carried out during the next generation of Arabidopsis thaliana plants, produced by agrobacterium transformation method. For this purpose two types of DNA-constructions were used for establishing better variant. The histochemical analysis of the plants progeny T1 transformed by both construct types was described. As a result of our work, it was established that the amount of marker-free transformants was arising during every next transformation offspring independently of the used construct type. The new strategy provides a simple and rapid way to eliminate swelective and marker genes.
- Published
- 2015
18. An Enhanced Gene Targeting Toolkit for Drosophila: Golic+
- Author
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Tzumin Lee, Barret D. Pfeiffer, Yaling Huang, Hui-Min Chen, and Xiaohao Yao
- Subjects
Genetics ,Cas9 ,Transgene ,Gene targeting ,DNA ,Investigations ,Biology ,Oogenesis ,Genome editing ,Gene Targeting ,Animals ,CRISPR ,Drosophila ,Female ,Site-specific recombinase technology ,Transgenes ,Homologous Recombination ,Homologous recombination ,Gene ,Ovum - Abstract
Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer “genes” in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.
- Published
- 2015
19. Adoption of the Q Transcriptional System for Regulating Gene Expression in Stem Cells
- Author
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Chelsea Gibbs, Michael Fitzgerald, Adrian A. Shimpi, and Tara L. Deans
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0301 basic medicine ,Transcription, Genetic ,Biomedical Engineering ,02 engineering and technology ,Computational biology ,CHO Cells ,Biology ,Lac repressor ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,03 medical and health sciences ,Synthetic biology ,Mice ,Cricetulus ,Gene expression ,Animals ,Humans ,Site-specific recombinase technology ,Gene ,Cell Engineering ,Genetics ,HEK 293 cells ,Mouse Embryonic Stem Cells ,General Medicine ,021001 nanoscience & nanotechnology ,Embryonic stem cell ,030104 developmental biology ,HEK293 Cells ,Gene Expression Regulation ,Stem cell ,0210 nano-technology - Abstract
The field of mammalian synthetic biology seeks to engineer enabling technologies to create novel approaches for programming cells to probe, perturb, and regulate gene expression with unprecedented precision. To accomplish this, new genetic parts continue to be identified that can be used to build novel genetic circuits to re-engineer cells to perform specific functions. Here, we establish a new transcription-based genetic circuit that combines genes from the quinic acid sensing metabolism of Neorospora crassa and the bacterial Lac repressor system to create a new orthogonal genetic tool to be used in mammalian cells. This work establishes a novel genetic tool, called LacQ, that functions to regulate gene expression in Chinese hamster ovarian (CHO) cells, human embryonic kidney 293 (HEK293) cells, and in mouse embryonic stem (ES) cells.
- Published
- 2017
20. Mechanistic basis for increased human gene targeting by promoterless vectors-roles of homology arms and Rad54 paralogs
- Author
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Aya Kurosawa, Shinta Saito, and Noritaka Adachi
- Subjects
0301 basic medicine ,DNA repair ,Genetic Vectors ,Biology ,Biochemistry ,Marker gene ,Homology (biology) ,Cell Line ,03 medical and health sciences ,Exon ,Gene Knockout Techniques ,Humans ,Site-specific recombinase technology ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Genetics ,DNA Helicases ,Gene targeting ,Nuclear Proteins ,Cell Biology ,Exons ,DNA-Binding Proteins ,030104 developmental biology ,Genetic Loci ,Homologous recombination - Abstract
Gene targeting by homologous recombination provides the definitive tool for analyzing gene function. Promoterless vectors, which do not possess a promoter to drive marker gene expression, confer higher targeting efficiencies than conventional vectors due to the reduced number of drug-resistant clones. We here show that gene-targeting efficiency is typically ≥ 25% with the use of exon-trapping-type promoterless vectors in a human diploid cell line, Nalm-6. The efficiency of exon-trapping gene targeting was correlated with the level of target gene expression when a 2A peptide sequence was linked to the marker gene. Intriguingly, total arm length was not necessarily a determinant of targeting efficiency, as longer arms tend to enhance both homologous (targeted) and nonhomologous (nontargeted) integration of the vector; rather, the presence of an exon in the 5' arm led to a decreased targeting efficiency. Strikingly, loss of Rad54 did not severely affect the targeting efficiency of exon-trap vectors. Moreover, additional deletion of the Rad54 paralog Rad54B had limited impact on the high-efficiency gene targeting. These results indicate that targeted integration occurs in human cells even when both Rad54 and Rad54B are missing. These studies provide additional important insight into the contribution of various DNA repair factors on the targeting mechanics.
- Published
- 2017
21. Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors
- Author
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David A. Sorrell, Linda Petrie, Andreas F. Kolb, Christopher Knowles, Claire Robinson, Jennifer A Harbottle, and Patrikas Pultinevicius
- Subjects
0301 basic medicine ,viruses ,Recombinase-mediated cassette exchange ,Computational biology ,Transfection ,Biology ,Genome ,Genome engineering ,Viral vector ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Recombinase ,Site-specific recombinase technology ,Gene ,030217 neurology & neurosurgery - Abstract
Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.
- Published
- 2017
22. Production of Minicircle DNA Vectors Using Site-Specific Recombinases
- Author
-
Kianoush Dormiani, Nafiseh Sanei Ata-abadi, Mohammad Hossein Nasr-Esfahani, and Naeimeh Rezaei
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,Plasmid ,Minicircle dna ,Genetic enhancement ,Transgene ,Recombinase ,Site-specific recombinase technology ,Computational biology ,Biology ,Minicircle ,Recombination - Abstract
Minicircle DNA vectors are plasmid derivatives free of bacterial elements. These vectors are mostly provided from common plasmids via recombination by site-specific recombinases in E. coli. Absence of bacterial backbone in minicircle vectors results in high-level and persistent expression of transgene in comparison with conventional plasmids and provides promising vehicles for gene therapy and vaccination. Here we describe the production of replicative minicircle DNA vectors using the PBAD/araC system expressing ΦC31 integrase in E. coli.
- Published
- 2017
23. Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments
- Author
-
Milena M. Awad, Jackie K. Cheung, Kate E. Mackin, Dena Lyras, Julian I. Rood, Glen P. Carter, Lauren Stevenson, Vicki Adams, Radhika Bantwal, and Joel Nicholson
- Subjects
DNA, Bacterial ,Clostridium perfringens ,Mutagenesis (molecular biology technique) ,Genetics and Molecular Biology ,Biology ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Recombinases ,Plasmid ,Bacterial Proteins ,Escherichia coli ,medicine ,Recombinase ,Site-specific recombinase technology ,Cloning, Molecular ,Gene ,Recombination, Genetic ,Genetics ,Mutation ,Ecology ,Genetic Complementation Test ,genomic DNA ,DNA Nucleotidyltransferases ,Genome, Bacterial ,Food Science ,Biotechnology - Abstract
TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn 4451 and Tn 4453 in Clostridium perfringens and Clostridium difficile , respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli . We show that TnpX represses expression from its own promoter, P attCI , which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens . Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.
- Published
- 2014
24. One-step transformation with site-specific recombinase Cre/loxP system under the control of minimal 35S promoter for the development of marker-free transgenicArabidopsis thalianaplants
- Author
-
S.V. Isaenkov and A.S. Sekan
- Subjects
Transformation (genetics) ,biology ,Transgene ,Arabidopsis thaliana ,Site-specific recombinase technology ,Cre-Lox recombination ,Marker free ,biology.organism_classification ,Floxing ,Cell biology - Published
- 2014
25. Gene Expression and Gene Ontology Enrichment Analysis for H3K4me3 and H3K4me1 in Mouse Liver and Mouse Embryonic Stem Cell Using ChIP-Seq and RNA-Seq
- Author
-
Ngoc Tam L. Tran and Chun-Hsi Huang
- Subjects
RNA-Seq ,Bioinformatics ,Genome ,03 medical and health sciences ,ChIP-Seq ,0302 clinical medicine ,Gene expression ,Genetics ,Medicine ,Site-specific recombinase technology ,Enhancer ,lcsh:QH301-705.5 ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Original Research ,0303 health sciences ,business.industry ,H3K4me1 ,H3K4me3 ,Embryonic stem cell ,Computer Science Applications ,Cell biology ,lcsh:Biology (General) ,030220 oncology & carcinogenesis ,gene expression ,gene ontology ,business - Abstract
Recent study has identified the cis-regulatory elements in the mouse genome as well as their genomic localizations. Recent discoveries have shown the enrichment of H3 lysine 4 trimethylation (H3K4me3) binding as an active promoter and the presence of H3 lysine 4 monomethylation (H3K4me1) outside promoter regions as a mark for an enhancer. In this work, we further identified highly expressed genes by H3K4me3 mark or by both H3K4me3 and H3K4me1 marks in mouse liver using ChIP-Seq and RNA-Seq. We found that in mice, the liver carries embryonic stem cell-related functions while the embryonic stem cell also carries liver-related functions. We also identified novel genes in RNA-Seq experiments for mouse liver and for mouse embryonic stem cells. These genes are not currently in the Ensemble gene database at NCBI.
- Published
- 2014
26. Host Organism:Streptomyces
- Author
-
Andriy Luzhetskyy and Oksana Bilyk
- Subjects
0301 basic medicine ,Genetics ,Reporter gene ,030106 microbiology ,Biology ,biology.organism_classification ,Streptomyces ,03 medical and health sciences ,030104 developmental biology ,Genomic engineering ,Host organism ,Transposon mutagenesis ,Site-specific recombinase technology ,Heterologous expression - Published
- 2016
27. GENE KICKED MOUSE: KNOCK OUT MOUSE AND ITS APPLICATION
- Author
-
Veterinary Dispensary, Rajashekar B, Suha sini K, and Pattar Jayashree
- Subjects
Genetically modified mouse ,Genetics ,Knockout mouse ,Laboratory mouse ,Cre recombinase ,Gene targeting ,Site-specific recombinase technology ,Biology ,Gene ,Embryonic stem cell - Abstract
A knockout mouse is a laboratory mouse in which genes are inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. The 2007 Nobel Prize in physiology or medicine is awarded to Drs Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of principles for introducing specific gene modifications in mice by using embryonic stem cells. Progress to gene targeting using embryonic cell was developed by Evans and his co - workers. Ingenious development of gene targeting has been made by introducing recognition sites for the enzyme Cre recombinase, called loxP sites, into existing genes. When mice carrying such "floxed" genes are mated with transgenic mice ex pressing Cre recombinase, the target gene of the offspring is modified through Cre action. Gene targeting has transformed scientific medicine by permitting experimental testing of hypotheses regarding the function of specific genes. The first area to which experim ental geneticists turned their attention after the birth of gene targeting in mammals was monogenic diseases. Gene targeting has been exceptionally useful in cancer research. A large number of protooncog enes, tumor suppressor genes, angiogenetic factors et c have been targeted in different tissues in mice to shed light on the induction and spreading of tumours. Gene - targeted mouse models have also become increasingly important in studies of host defense against pathogens. Gene targeted mice have become i ndis pensable in virtually all aspects of medical research.
- Published
- 2013
28. Nanos3 Gene Targeting in Medaka ES Cells
- Author
-
Guijun Guan, Yoshitaka Nagahama, Hong Ni, Kiyoshi Naruse, Tiansheng Chen, Yunhan Hong, Yan Yan, and Meisheng Yi
- Subjects
Pluripotent Stem Cells ,Genotype ,Genetic Vectors ,Oryzias ,homologous recombination ,Biology ,nanos3 ,Applied Microbiology and Biotechnology ,gene targeting ,Mice ,Species Specificity ,Animals ,Site-specific recombinase technology ,pluripotency ,Molecular Biology ,Gene ,Zebrafish ,Ecology, Evolution, Behavior and Systematics ,Embryonic Stem Cells ,Reverse Transcriptase Polymerase Chain Reaction ,Gene targeting ,RNA-Binding Proteins ,Cell Biology ,Sequence Analysis, DNA ,biology.organism_classification ,Molecular biology ,Embryonic stem cell ,Blotting, Southern ,ES ,NANOS3 Gene ,Stem cell ,Homologous recombination ,Genetic Engineering ,Developmental Biology ,Research Paper ,Plasmids - Abstract
Gene targeting (GT) by homologous recombination offers the best precision for genome editing in mice. nanos3 is a highly conserved gene and encodes a zinc-finger RNA binding protein essential for germ stem cell maintenance in Drosophila, zebrafish and mouse. Here we report nanos3 GT in embryonic stem (ES) cells of the fish medaka as a lower vertebrate model organism. A vector was designed for GT via homologous recombination on the basis of positive-negative selection (PNS). The ES cell line MES1 after gene transfer and PNS produced 56 colonies that were expanded into ES cell sublines. Nine sublines were GT-positive by PCR genotyping, 4 of which were homologous recombinants as revealed by Southern blot. We show that one of the 4, A15, contains a precisely targeted nanos3 allele without any random events, demonstrating the GT feasibility in medaka ES cells. Importantly, A15 retained all features of undifferentiated ES cells, including stable self-renewal, an undifferentiated phenotype, pluripotency gene expression and differentiation during chimeric embryogenesis. These results provide first evidence that the GT procedure and genuine GT on a chromosomal locus such as nanos3 do not compromise pluripotency in ES cells of a lower vertebrate.
- Published
- 2013
29. A new site-specific recombinase-mediated system for targeted multiple genomic deletions employing chimeric loxP and mrpS sites
- Author
-
Josef Altenbuchner and Lydia Warth
- Subjects
Recombination, Genetic ,Genetics ,Base Sequence ,Integrases ,Cre recombinase ,Genomics ,General Medicine ,Chromosomes, Bacterial ,Biology ,Polymerase Chain Reaction ,Applied Microbiology and Biotechnology ,Plasmid ,Sequence Homology, Nucleic Acid ,Escherichia coli ,Recombinase ,Site-specific recombinase technology ,Cre-Lox recombination ,Site-specific recombination ,Binding site ,Gene Deletion ,Floxing ,Plasmids ,Biotechnology - Abstract
A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.
- Published
- 2013
30. Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering
- Author
-
M. Teresa Pisabarro, Victoria Splith, Madina Karimova, Janet Karpinski, and Frank Buchholz
- Subjects
0301 basic medicine ,Biology ,Genome ,Article ,Genome engineering ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Plasmid ,Recombinase ,Animals ,Humans ,Site-specific recombinase technology ,DNA Nucleotidyltransferases ,Genetics ,Recombination, Genetic ,Multidisciplinary ,DNA ,genomic DNA ,030104 developmental biology ,chemistry ,Tyrosine ,Genetic Engineering ,030217 neurology & neurosurgery ,Plasmids - Abstract
Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets.
- Published
- 2016
31. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?
- Author
-
Robert Gerlai
- Subjects
0301 basic medicine ,Genetics ,Transcription activator-like effector nuclease ,lcsh:QH426-470 ,Cas9 ,Gene targeting ,Review ,Geneticist ,Computational biology ,Biology ,embryonic stem cell ,lcsh:Genetics ,03 medical and health sciences ,030104 developmental biology ,Gene Targeting ,Molecular Medicine ,CRISPR ,Site-specific recombinase technology ,Behavior Genetics ,Homologous Recombination ,Homologous recombination ,Genetics (clinical) ,Behavioural genetics ,Neuroscience - Abstract
Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.
- Published
- 2016
32. Vegfr3-CreER (T2) mouse, a new genetic tool for targeting the lymphatic system
- Author
-
Rodrigo Diéguez-Hurtado, Lukas Stanczuk, Ines Martinez-Corral, Sagrario Ortega, Taija Makinen, Maria H. Ulvmar, David Olmeda, and Maike Frye
- Subjects
0301 basic medicine ,Cancer Research ,Mice, 129 Strain ,Physiology ,Angiogenesis ,government.form_of_government ,Clinical Biochemistry ,Cre recombinase ,Mice, Transgenic ,Biology ,Lymphatic System ,Mice ,03 medical and health sciences ,Pregnancy ,Recombinase ,Animals ,Site-specific recombinase technology ,Cardiac and Cardiovascular Systems ,Gene Knock-In Techniques ,Kardiologi ,Integrases ,Gene Expression Regulation, Developmental ,Gene targeting ,Vascular Endothelial Growth Factor Receptor-3 ,Embryonic stem cell ,Molecular biology ,Cell biology ,Mice, Inbred C57BL ,Tamoxifen ,Lymphatic Endothelium ,030104 developmental biology ,Lymphatic system ,government ,Female - Abstract
The lymphatic system is essential in many physiological and pathological processes. Still, much remains to be known about the molecular mechanisms that control its development and function and how to modulate them therapeutically. The study of these mechanisms will benefit from better controlled genetic mouse models targeting specifically lymphatic endothelial cells. Among the genes expressed predominantly in lymphatic endothelium, Vegfr3 was the first one identified and is still considered to be one of the best lymphatic markers and a key regulator of the lymphatic system. Here, we report the generation of a Vegfr3-CreER (T2) knockin mouse by gene targeting in embryonic stem cells. This mouse expresses the tamoxifen-inducible CreER(T2) recombinase under the endogenous transcriptional control of the Vegfr3 gene without altering its physiological expression or regulation. The Vegfr3-CreER (T2) allele drives efficient recombination of floxed sequences upon tamoxifen administration specifically in Vegfr3-expressing cells, both in vitro, in primary lymphatic endothelial cells, and in vivo, at different stages of mouse embryonic development and postnatal life. Thus, our Vegfr3-CreER (T2) mouse constitutes a new powerful genetic tool for lineage tracing analysis and for conditional gene manipulation in the lymphatic endothelium that will contribute to improve our current understanding of this system. Erratum in: Angiogenesis 19(3) p. 447 DOI: 10.1007/s10456-016-9518-5
- Published
- 2016
33. Actinomycetes genome engineering approaches
- Author
-
Andriy Luzhetskyy and Theresa Siegl
- Subjects
Genetics, Microbial ,Transposable element ,Genetics ,General Medicine ,Biology ,Microbiology ,Genome ,Genome engineering ,Actinobacteria ,Recombinase ,Site-specific recombinase technology ,Transposon mutagenesis ,Genetic Engineering ,Molecular Biology ,Gene ,Genome, Bacterial ,Function (biology) - Abstract
This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA and I-SceI systems). We will describe some new vectors recently developed for engineering of complex phenotypes in actinomycetes. Several site-specific recombinases, transposons, reporter genes and I-SceI endonuclease have been utilized for genome manipulation in actinomycetes. Novel molecular tools will help to overcome many technical difficulties and will encourage new efforts to address the function of actinomycete genes.
- Published
- 2012
34. Conditional gene manipulation: Cre-ating a new biological era
- Author
-
Jing Zhao, Wen-jie Jiang, Xi-wei Shan, Xiao-mei Yang, Jian Zhang, and Jiangang Gao
- Subjects
Genetics ,General Veterinary ,Genome, Human ,Transgene ,food and beverages ,Gene targeting ,General Medicine ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Recombinases ,Gene trapping ,Gene Targeting ,Recombinase ,Humans ,Gene silencing ,Site-specific recombinase technology ,Gene Silencing ,General Pharmacology, Toxicology and Pharmaceutics ,Genetic Engineering ,Gene ,Gene knockout ,Biotechnology - Abstract
To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase (SSR) systems (Cre-loxP, Flp-FRT, and ΦC31) have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems (tetracycline and tamoxifen), stage-specific knockout and transgenic expression can be achieved. The application of this “SSR+inducible” conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.
- Published
- 2012
35. Rapid and Cost-Effective Gene Targeting in Rat Embryonic Stem Cells by TALENs
- Author
-
Qi-Long Ying, Charles Ashton, Guanyi Huang, Hong-Ping Wu, Chang Tong, and He-Xin Yan
- Subjects
Recombination, Genetic ,Transcription activator-like effector nuclease ,Knockout rat ,Base Sequence ,Genetic Vectors ,Molecular Sequence Data ,Gene targeting ,Transfection ,Biology ,Molecular biology ,Embryonic stem cell ,Article ,Cell Line ,Rats ,Mice ,TAL effector ,Gene Order ,Gene Targeting ,Genetics ,Animals ,Site-specific recombinase technology ,Homologous recombination ,Molecular Biology ,Embryonic Stem Cells - Abstract
The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.
- Published
- 2012
36. Challenges to increasing targeting efficiency in genome engineering
- Author
-
Izuho Hatada and Takuro Horii
- Subjects
0301 basic medicine ,Knockout ,Biology ,Gene dosage ,Genome engineering ,03 medical and health sciences ,Mice ,Genome editing ,CRISPR ,Animals ,Humans ,Site-specific recombinase technology ,Homologous recombination ,Gene knockout ,Embryonic Stem Cells ,Genetics ,Gene Editing ,Mice, Knockout ,Genome ,Models, Genetic ,Cell Cycle ,Gene targeting ,030104 developmental biology ,Knockin ,Mutation ,Animal Science and Zoology ,CRISPR-Cas Systems ,Opinions and Hypotheses ,Genetic Engineering - Abstract
Gene targeting technologies are essential for the analysis of gene functions. Knockout mouse generation via genetic modification of embryonic stem cells (ESCs) is the commonest example, but it is a time-consuming and labor-intensive procedure. Recently, a novel genome editing technology called CRISPR/Cas has enabled the direct production of knockout mice by non-homologous end joining (NHEJ)-mediated mutations. Unexpectedly, however, it generally exhibits a low efficiency in homologous recombination (HR) and is prone to high mosaicism. Meanwhile, gene targeting using ESCs is still being improved, as reported by Fukuda et al. in this issue. Here, we outline current gene targeting technologies with special emphasis on HR-mediated technologies, which are currently being performed using these two major strategies.
- Published
- 2015
37. Site-Specific Recombinase Strategy to Create Induced Pluripotent Stem Cells Efficiently with Plasmid DNA
- Author
-
Christopher L. Chavez, Joseph C. Wu, Jonathan M. Geisinger, Alfonso P. Farruggio, Marisa Karow, Yanru Chen-Tsai, Michele P. Calos, Feng Lan, W. Edward Jung, and Annahita Keravala
- Subjects
Genetics ,Mesenchymal stem cell ,Cre recombinase ,Cell Biology ,Transfection ,Biology ,Cell biology ,Recombinase ,Molecular Medicine ,Site-specific recombinase technology ,Stem cell ,Induced pluripotent stem cell ,Reprogramming ,Developmental Biology - Abstract
Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.
- Published
- 2011
38. The Gene or Not the Gene—That Is the Question
- Author
-
Rani S. Sellers
- Subjects
Genetics ,Time Factors ,General Veterinary ,Gene Expression ,Genetic Variation ,Gene targeting ,Tissue-Specific Gene Expression ,Mice, Transgenic ,Biology ,Embryonic stem cell ,Phenotype ,Mice ,Organ Specificity ,Genetically Engineered Mouse ,Mutation ,Gene expression ,Animals ,Site-specific recombinase technology ,Transgenes ,Genetic Engineering ,Gene ,Alleles ,Embryonic Stem Cells - Abstract
Embryonic stem cells have had a significant impact on understanding gene function and gene interactions through the use of genetically engineered mice. However, the genetic context (ie, mouse strain) in which these modifications in alleles are made may have a considerable effect on the phenotypic changes identified in these mice. In addition, tissue- and time-specific gene expression systems may generate unanticipated outcomes. This article discusses the history of embryonic stem cells, reviews how mouse strain can affect phenotype (using specific examples), and examines some of the caveats of conditional gene expression systems.
- Published
- 2011
39. Homologous Recombination in Human Embryonic Stem Cells: A Tool for Advancing Cell Therapy and Understanding and Treating Human Disease
- Author
-
Andrew D. Leavitt and Isla Hamlett
- Subjects
Genetics ,General Neuroscience ,Recombinase-mediated cassette exchange ,Gene targeting ,General Medicine ,Disease ,Computational biology ,Biology ,Embryonic stem cell ,General Biochemistry, Genetics and Molecular Biology ,Cell therapy ,Gene Targeting ,Recombinase ,Humans ,Site-specific recombinase technology ,General Pharmacology, Toxicology and Pharmaceutics ,Homologous Recombination ,Homologous recombination ,Research Articles ,Embryonic Stem Cells ,Stem Cell Transplantation - Abstract
Human embryonic stem cells (hESCs) hold great promise for ushering in an era of novel cell therapies to treat a wide range of rare and common diseases, yet they also provide an unprecedented opportunity for basic research to yield clinical benefit. HESCs can be used to better understand human development, to model human diseases, to understand the contribution of specific mutations to the pathogenesis of disease, and to develop human cell‐based screening systems to identify novel therapeutic agents and evaluate potential toxicity of therapeutic agents under development. Such basic research will benefit greatly from efficient methods to perform targeted gene modification, an area of hESC investigation that is currently in its infancy. Moreover, the reality of hESC‐based cellular therapies will require improved methods for generating the specific cells of interest, and reporter cell lines generated through targeted gene modifications are expected to play an important role in developing optimal cell‐specific differentiation protocols. Herein, we review the current status of homologous recombination in hESCs, a gene targeting technique that is sure to continue to improve, and to play an important role in realizing the maximal human benefit from hESCs. Clin Trans Sci 2011; Volume 4: 298–305
- Published
- 2011
40. Drug-Inducible Gene Recombination by the Dppa3-MER Cre MER Transgene in the Developmental Cycle of the Germ Cell Lineage in Mice1
- Author
-
Mayo Shigeta, Mitinori Saitou, Hitoshi Niwa, Hiroshi Ohta, and Takayuki Hirota
- Subjects
Genetics ,Regulation of gene expression ,Transgene ,Cre recombinase ,Cell Biology ,General Medicine ,Biology ,C-Mer Tyrosine Kinase ,medicine.anatomical_structure ,Reproductive Medicine ,medicine ,Site-specific recombinase technology ,Gene ,Germ cell ,Floxing - Abstract
Germ cells ensure the diversification and totipotency of genetic information via the elaborate genetic and epigenetic regulation of the genome architecture during their development. To understand the mechanism underlying the regulation of genome function in germ cells, it is of primary importance to develop systems in which gene function can be regulated at desired time points during their development. Here, we report the generation of transgenic strains that express Cre recombinase flanked by the ligand-binding domains of murine estrogen receptor (MER Cre MER [MCM]) under the control of the regulatory elements of the Dppa3 (also known as Stella or Pgc7) gene. On the administration of 4-hydroxytamoxifen (4-OHT), the Dppa3-MCM strains recombined the sequence flanked by the loxP elements (the floxed sequence) specifically in primordial germ cells as early as Embryonic Day (E) 7.0, and this recombination became robust after E9.5. Furthermore, these strains exhibited efficient and specific recombination of the floxed sequence during the growth of oocytes and in preimplantation embryos in the 4-OHT-dependent manner. Thus, these Dppa3-MCM strains offer valuable opportunities to explore gene function in both loss-of-function and gain-of-function experiments at a variety of time points during germ cell development.
- Published
- 2011
41. Mouse mutants and phenotypes: Accessing information for the study of mammalian gene function
- Author
-
Martin Ringwald and Janan T. Eppig
- Subjects
Mutant ,Biology ,medicine.disease_cause ,Online Systems ,Genome ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Recombinases ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Databases, Genetic ,medicine ,Recombinase ,Animals ,Humans ,Site-specific recombinase technology ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Embryonic Stem Cells ,030304 developmental biology ,Genetics ,0303 health sciences ,Mutation ,Genetic Diseases, Inborn ,Gene targeting ,Phenotype ,Mice, Mutant Strains ,Disease Models, Animal ,Genes ,Organ Specificity ,030217 neurology & neurosurgery - Abstract
Recent advances in high-throughput gene targeting and conditional mutagenesis are creating new and powerful resources to study the in-vivo function of mammalian genes using the mouse as an experimental model. Mutant ES cells and mice are being generated at a rapid rate to study the molecular and phenotypic consequences of genetic mutations, and to correlate these study results with human disease conditions. Likewise, classical genetics approaches to identify mutations in the mouse genome that cause specific phenotypes have become more effective. Here, we describe methods to quickly obtain information on what mutant ES cells and mice are available, including recombinase driver lines for the generation of conditional mutants. Further, we describe means to access genetic and phenotypic data that identify mouse models for specific human diseases.
- Published
- 2011
42. Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges
- Author
-
Juergen Bode, Christoph Zehe, Christine Voelkel, Ellen Ernst, Melanie Galla, Bernhard Schiedlmeier, Soeren Turan, and Junhua Qiao
- Subjects
Genetics ,biology ,Recombinase-mediated cassette exchange ,Transgene ,Gene Transfer Techniques ,Integrase ,Mice ,Transduction (genetics) ,Gene trapping ,Structural Biology ,DNA Nucleotidyltransferases ,Gene Targeting ,biology.protein ,Recombinase ,Animals ,Site-specific recombinase technology ,Transgenes ,Genetic Engineering ,Molecular Biology ,Gene - Abstract
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.
- Published
- 2011
43. Site-specific integration of transgene targeting an endogenous lox-like site in early mouse embryos
- Author
-
Michele P. Calos, Masanori Ito, Hideaki Tojo, Keitaro Yamanouchi, and Kunihiko Naito
- Subjects
Male ,Transgene ,Cre recombinase ,Endogeny ,Biology ,Animals, Genetically Modified ,Mice ,Plasmid ,Genetics ,Animals ,Site-specific recombinase technology ,Transgenes ,Gene ,Recombination, Genetic ,Mice, Inbred C3H ,Mice, Inbred ICR ,Integrases ,General Medicine ,Embryo, Mammalian ,beta-Galactosidase ,Molecular biology ,Mice, Inbred C57BL ,Transgenesis ,Lac Operon ,Gene Targeting ,Female ,Exogenous DNA ,Plasmids - Abstract
Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (ψloxm5) in early mouse embryos. We characterized the Cre/ψloxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ψloxm5 and ψloxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ψloxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ψloxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/ψloxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.
- Published
- 2010
44. ROSA26Flpo deleter mice promote efficient inversion of conditional gene traps in vivo
- Author
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Christopher S. Raymond and Philippe Soriano
- Subjects
RNA, Untranslated ,FLP-FRT recombination ,Genetic Vectors ,Mutagenesis (molecular biology technique) ,Mice, Transgenic ,Computational biology ,Biology ,Article ,Recombinases ,Mice ,Endocrinology ,Plasmid ,Genes, Reporter ,In vivo ,Genetics ,Recombinase ,Animals ,Site-specific recombinase technology ,Gene ,Embryonic Stem Cells ,Proteins ,DNA ,Cell Biology ,Embryonic stem cell ,Mutagenesis, Insertional ,Retroviridae ,Chromosome Inversion ,DNA Nucleotidyltransferases ,Gene Deletion ,Plasmids - Abstract
Gene trap mutagenesis in embryonic stem (ES) cells is an important tool to help elucidate gene function in current mouse mutagenesis efforts. Vector systems based on inversion of the gene trap module have recently been devised to allow for conditional mutagenesis. However, additional efforts are needed to improve this technology including improving the efficiency of site-specific recombinases required to manipulate these conditional vectors in vivo. Here we describe a mouse line carrying the codon-optimized FLP recombinase Flpo at the ROSA26 locus that functions at higher efficiency than a similar Flpe line in mediating the DNA inversion of a conditional gene trap cassette in vivo.
- Published
- 2010
45. Applications of the site-specific recombinase Cre to the study of genomic imprinting
- Author
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Louis Lefebvre, Meaghan J. Jones, and Rosemary Oh-McGinnis
- Subjects
Genetics ,Chromosome engineering ,Integrases ,Models, Genetic ,Cre recombinase ,General Medicine ,Chromosomal rearrangement ,Biology ,Biochemistry ,Article ,Genomic Imprinting ,Mice ,DNA Nucleotidyltransferases ,Recombinase ,Animals ,Humans ,Site-specific recombinase technology ,Imprinting (psychology) ,Genomic imprinting ,Molecular Biology ,Floxing - Abstract
The development of gene targeting approaches has had a tremendous impact on the functional analysis of the mouse genome. A specific application of this technique has been the adaptation of the bacteriophage P1 Cre/loxP site-specific recombinase system which allows for the precise recombination between two loxP sites, resulting in deletion or inversion of the intervening sequences. Because of the efficiency of this system, it can be applied to conditional deletions of relatively short coding sequences or regulatory elements but also to more extensive chromosomal rearrangement strategies. Both mechanistic and functional studies of genomic imprinting have benefited from the development of the Cre/loxP technology. Since imprinted genes within large chromosomal regions are regulated by the action of cis-acting sequences known as imprinting centres, chromosomal engineering approaches are particularly well suited to the elucidation of long-range mechanisms controlling the imprinting of autosomal genes. Here we review the applications of the Cre/loxP technology to the study of genomic imprinting, highlight important insights gained from these studies and discuss future directions in the field.
- Published
- 2010
46. Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision
- Author
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Howard A. Baylis, Rafael P. Vázquez-Manrique, Sung Ly, Birgitta Olofsson, and James C. Legg
- Subjects
Genetic Markers ,FLP-FRT recombination ,Green Fluorescent Proteins ,Gene Expression ,Nerve Tissue Proteins ,Computational biology ,Marker gene ,Gene knockout ,Animals, Genetically Modified ,Gene Knockout Techniques ,Flp recombinase ,Phospholipase C ,Conditional gene knockout ,Genetics ,Animals ,Site-specific recombinase technology ,Homologous recombination ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Genes, Helminth ,Biolistic transformation ,Recombination, Genetic ,Microscopy, Confocal ,biology ,Gene targeting ,biology.organism_classification ,Microscopy, Fluorescence ,DNA Nucleotidyltransferases ,Phospholipase C delta - Abstract
Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-δ in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.
- Published
- 2010
47. Characterization of transgenic mouse lines expressing Cre recombinase in the retina
- Author
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Grace-Soon Hwang, Elena Ivanova, and Zhuo-Hua Pan
- Subjects
Cell type ,Retina ,Integrases ,General Neuroscience ,EMX1 ,Cre recombinase ,Mice, Transgenic ,Biology ,Retinal ganglion ,Molecular biology ,Recombinant Proteins ,Article ,Amacrine cell ,Mice ,medicine.anatomical_structure ,medicine ,Animals ,Ectopic expression ,Site-specific recombinase technology ,sense organs - Abstract
The mammalian retina consists of five major classes of neuronal cells, as well as glial cells, and it contains more than 50 cell types. The ability to manipulate gene expression in specific cell type(s) in the retina is important for understanding the molecular mechanisms of retinal function and diseases. The Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in vivo in a cell- and tissue-specific fashion. The key to this tool is the availability of Cre mouse lines with cell- or tissue-type specific expression of Cre recombinase. To date, a large number of Cre-transgenic mouse lines have been generated to target Cre recombinase expression to specific neuronal and glial cell populations in the central nervous system; however, information about the expression patterns of Cre recombinase lines in the retina is largely lacking. In this study, we examined and characterized the expression patterns of Cre recombinase in the retinas of 15 Cre-transgenic mouse lines. Significant Cre-induced recombination or expression of Cre recombinase was observed in the majority of these lines. In particular, we found several Cre lines in which the Cre-induced recombination was found to target exclusively or predominantly a single type or class of retinal cells, including bistratified retinal ganglion cells, starburst amacrine cells, rod bipolar cells, and Müller glial cells. In other lines, the Cre-induced recombination was found in several retinal cell types. These Cre lines provide a valuable resource for retinal research.
- Published
- 2010
48. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells
- Author
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Yaobin Liu, Rodney S. Nairn, and Karen M. Vasquez
- Subjects
Gene Conversion ,Oligonucleotides ,Genome Integrity, Repair and Replication ,Biology ,Polymerase Chain Reaction ,03 medical and health sciences ,chemistry.chemical_compound ,Plasmid ,Genetics ,Humans ,Site-specific recombinase technology ,Gene conversion ,Gene ,030304 developmental biology ,Recombination, Genetic ,0303 health sciences ,Oligonucleotide ,030302 biochemistry & molecular biology ,Ficusin ,Gene targeting ,DNA ,Molecular biology ,Cross-Linking Reagents ,chemistry ,Homologous recombination ,Gene Deletion ,HeLa Cells - Abstract
Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.
- Published
- 2009
49. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors
- Author
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Kaoru Mitsui, Kohnosuke Mitani, Hirofumi Suemori, Keiichiro Suzuki, Toshiyuki Yamagishi, Norio Nakatsuji, Kouichi Hasegawa, Yoshihiko Shimizu, Eihachiro Kawase, and Emi Aizawa
- Subjects
Hypoxanthine Phosphoribosyltransferase ,Multidisciplinary ,Genetic Vectors ,Gene Transfer Techniques ,Gene Expression ,Gene targeting ,Biological Sciences ,Gene delivery ,Biology ,Embryonic stem cell ,Molecular biology ,Adenoviridae ,Cell Line ,Macaca fascicularis ,Mice ,Cell culture ,Gene Targeting ,Animals ,Humans ,Site-specific recombinase technology ,Stem cell ,Homologous recombination ,Induced pluripotent stem cell ,Embryonic Stem Cells - Abstract
Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey ( Macaca fasicularis ) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.
- Published
- 2008
50. Marker removal from actinomycetes genome using Flp recombinase
- Author
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Lutz Petzke, Andreas Bechthold, Marta Fedoryshyn, Elisabeth Welle, and Andriy Luzhetskyy
- Subjects
Genetic Markers ,FLP-FRT recombination ,Gene Expression ,Streptomyces coelicolor ,Apramycin ,Streptomyces ,Actinomycetales ,Genes, Synthetic ,Genetics ,medicine ,Recombinase ,Nebramycin ,Site-specific recombinase technology ,Gene ,Recombination, Genetic ,biology ,Drug Resistance, Microbial ,General Medicine ,biology.organism_classification ,Molecular biology ,DNA Nucleotidyltransferases ,Streptomyces lividans ,Chromosomes, Fungal ,Genome, Fungal ,Genetic Engineering ,GC-content ,medicine.drug - Abstract
We report here a system for the functional expression of the Flp recombinase in several actinomycetes: Streptomyces coelicolor, S. lividans, and Saccharotrix espanaensis. We have constructed a synthetic gene encoding the Flp recombinase with a GC content of 60.6% optimized for expression in high-GC bacteria. Using the synthetic flp(a) gene, we have removed an apramycin resistance gene flanked by FRT sites from the chromosome of actinomycetes with an efficiency of 40%. Sequencing the region of chromosome showed that excision of the apramycin cassette by Flp recombinase was specific.
- Published
- 2008
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