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Pancreas-specific Cre driver lines and considerations for their prudent use.
Magnuson MA, Osipovich AB
(2013) Cell Metab 18: 9-20
MeSH Terms: Animals, DNA Nucleotidyltransferases, Extracellular Matrix Proteins, Integrases, Mice, Mice, Knockout, Mice, Transgenic, Models, Animal, Pancreas, Pancreatic Hormones, Protein-Lysine 6-Oxidase, Rats, Rats, Transgenic, Recombination, Genetic, Transgenes
Show Abstract · Added August 1, 2013
Cre/LoxP has broad utility for studying the function, development, and oncogenic transformation of pancreatic cells in mice. Here we provide an overview of the Cre driver lines that are available for such studies. We discuss how variegated expression, transgene silencing, and recombination in undesired cell types have conspired to limit the performance of these lines, sometimes leading to serious experimental concerns. We also discuss preferred strategies for achieving high-fidelity driver lines and remind investigators of the continuing need for caution when interpreting results obtained from any Cre/LoxP-based experiment performed in mice.
Copyright © 2013 Elsevier Inc. All rights reserved.
2 Communities
2 Members
0 Resources
15 MeSH Terms
Conditional control of gene function by an invertible gene trap in zebrafish.
Ni TT, Lu J, Zhu M, Maddison LA, Boyd KL, Huskey L, Ju B, Hesselson D, Zhong TP, Page-McCaw PS, Stainier DY, Chen W
(2012) Proc Natl Acad Sci U S A 109: 15389-94
MeSH Terms: Alleles, Animals, DNA Nucleotidyltransferases, DNA Transposable Elements, Hepatocytes, Integrases, Liver, Mitochondria, Models, Genetic, Mutagenesis, Mutagens, Mutation, Phenotype, Polymerase Chain Reaction, RNA Helicases, Recombination, Genetic, Zebrafish
Show Abstract · Added January 7, 2014
Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.
1 Communities
3 Members
0 Resources
17 MeSH Terms
Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background.
Birling MC, Dierich A, Jacquot S, Hérault Y, Pavlovic G
(2012) Genesis 50: 482-9
MeSH Terms: Animals, Bacterial Proteins, DNA Nucleotidyltransferases, Founder Effect, Gene Targeting, Genetic Engineering, Genotype, Green Fluorescent Proteins, Homologous Recombination, Integrases, Luminescent Proteins, Mice, Mice, Inbred C57BL, Mice, Transgenic, Proteins, RNA, Untranslated, Sequence Deletion
Show Abstract · Added March 20, 2013
To facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker, respectively the eGFP or the eYFP, and were inserted by homologous recombination in the neutral Rosa26 locus. They allow a rapid, cost-effective, and efficient identification of the carrier individuals through the coexpression of the fluorescent marker. The recombination efficiency of the two deleter lines, Gt(ROSA)26S or < tm1(ACTB-cre,-EGFP)Ics> and Gt(ROSA) 26S or < tm2(CAG-flpo, EYFP)Ics>, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each tested locus, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains easily detectable until 10 days after birth. In the adult, fluorescence can still be detected in the unpigmented paws. Furthermore, they both display accumulation of the specific recombinase during oogenesis. These fluorescent 'Cre- and Flp- deleter' transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the maintenance of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.
Copyright © 2012 Wiley Periodicals, Inc.
1 Communities
0 Members
0 Resources
17 MeSH Terms
An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase.
Kranz A, Fu J, Duerschke K, Weidlich S, Naumann R, Stewart AF, Anastassiadis K
(2010) Genesis 48: 512-20
MeSH Terms: Animals, Base Sequence, Binding Sites, Cell Line, Cells, Cultured, DNA Nucleotidyltransferases, Embryo, Mammalian, Embryonic Stem Cells, Female, Fibroblasts, Gene Expression Regulation, Developmental, Gene Knockout Techniques, Green Fluorescent Proteins, Isoenzymes, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Plasmids, Recombination, Genetic, Transfection
Show Abstract · Added March 20, 2013
Recently, a codon improved version of the Flpe site specific recombinase, termed Flpo, was reported as having greatly improved performance in mammalian cell applications. However, the degree of improvement could not be estimated because essentially no Flpe activity was observed. Here, we compare Flpe and Flpo accurately in a mammalian cell assay to estimate that Flpo is about five times more active than Flpe and similar to Cre and Dre. Consequently, we generated a Flpo deleter mouse line from the JM8 C57Bl/6 ES cells used in the EUCOMM and KOMP systematic knock-out programs. In breeding experiments, we show that the Flpo deleter delivers complete recombination using alleles that are incompletely recombined by a commonly used Flpe deleter. This indicates that the Flpo deleter is more efficient.
(c) 2010 Wiley-Liss, Inc.
1 Communities
0 Members
0 Resources
21 MeSH Terms
Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.
Anastassiadis K, Fu J, Patsch C, Hu S, Weidlich S, Duerschke K, Buchholz F, Edenhofer F, Stewart AF
(2009) Dis Model Mech 2: 508-15
MeSH Terms: Animals, Base Sequence, Cell Line, DNA Nucleotidyltransferases, Embryonic Stem Cells, Escherichia coli, Escherichia coli Proteins, Gene Targeting, Genes, Reporter, Integrases, Ligands, Mice, Mifepristone, Molecular Sequence Data, Plasmids, Progesterone, Prokaryotic Cells, Proteins, RNA, Untranslated, Recombinant Fusion Proteins, Recombinases, Recombination, Genetic, Reproducibility of Results
Show Abstract · Added June 25, 2010
Tyrosine site-specific recombinases (SSRs) including Cre and FLP are essential tools for DNA and genome engineering. Cre has long been recognized as the best SSR for genome engineering, particularly in mice. Obtaining another SSR that is as good as Cre will be a valuable addition to the genomic toolbox. To this end, we have developed and validated reagents for the Dre-rox system. These include an Escherichia coli-inducible expression vector based on the temperature-sensitive pSC101 plasmid, a mammalian expression vector based on the CAGGs promoter, a rox-lacZ reporter embryonic stem (ES) cell line based on targeting at the Rosa26 locus, the accompanying Rosa26-rox reporter mouse line, and a CAGGs-Dre deleter mouse line. We also show that a Dre-progesterone receptor shows good ligand-responsive induction properties. Furthermore, we show that there is no crossover recombination between Cre-rox or Dre-loxP. Hence, we add another set of efficient tools to the genomic toolbox, which will enable the development of more sophisticated mouse models for the analysis of gene function and disease.
1 Communities
0 Members
1 Resources
23 MeSH Terms
FlEx-based transgenic reporter lines for visualization of Cre and Flp activity in live zebrafish.
Boniface EJ, Lu J, Victoroff T, Zhu M, Chen W
(2009) Genesis 47: 484-91
MeSH Terms: Animals, Animals, Genetically Modified, Autophagy, Base Sequence, DNA Nucleotidyltransferases, DNA Primers, Genes, Reporter, Integrases, Polymerase Chain Reaction, Zebrafish
Show Abstract · Added April 24, 2014
Site-specific recombinases such as Cre and Flp are invaluable tools for genetic manipulations, but their usage in zebrafish has been limited. Incorporating recently developed flip-excision (FlEx) design that allows stable inversions, we have established zebrafish reporter lines that express bright and ubiquitous EGFP, but switch to express mCherry in the presence of Cre or Flp. Here, we demonstrate the stable inversion in the reporter lines, both in somatic cells and in the germ line by Cre or Flp, and the subsequent reinversion using the other recombinase. Using the reporter lines, we characterized cardiomyocyte-specific Cre lines and neuronal progenitor-specific and tamoxifen-dependent Cre lines. We also used the reporter lines for screening Cre- and Flp-based enhancer trap lines. Similar to the widely used Cre reporter lines in mice, these FlEx-based reporter lines will facilitate the use of recombinases for genetic manipulations in zebrafish.
2009 Wiley-Liss, Inc.
0 Communities
1 Members
0 Resources
10 MeSH Terms
High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells.
Raymond CS, Soriano P
(2007) PLoS One 2: e162
MeSH Terms: Animals, DNA Nucleotidyltransferases, Embryo, Mammalian, Genes, Reporter, Genetic Engineering, Integrases, Mice, Mice, Transgenic, Recombination, Genetic
Show Abstract · Added March 20, 2013
DNA site-specific recombinases (SSRs) such as Cre, FLPe, and phiC31, are powerful tools for analyzing gene function in vertebrates. While the availability of multiple high-efficiency SSRs would facilitate a wide array of genomic engineering possibilities, efficient recombination in mammalian cells has only been observed with Cre recombinase. Here we report the de novo synthesis of mouse codon-optimized FLP (FLPo) and PhiC31 (PhiC31o) SSRs, which result in recombination efficiencies similar to Cre.
1 Communities
0 Members
1 Resources
9 MeSH Terms
High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP.
Rodríguez CI, Buchholz F, Galloway J, Sequerra R, Kasper J, Ayala R, Stewart AF, Dymecki SM
(2000) Nat Genet 25: 139-40
MeSH Terms: Amino Acid Substitution, Animals, DNA Nucleotidyltransferases, Enzyme Stability, Female, Genes, Reporter, Genetic Engineering, Integrases, Male, Mice, Mice, Transgenic, Mutagenesis, Site-Directed, Organ Specificity, Recombination, Genetic, Sequence Deletion, Transgenes, Viral Proteins
Added July 18, 2013
0 Communities
0 Members
1 Resources
17 MeSH Terms
Failure of hairpin-ended and nicked DNA To activate DNA-dependent protein kinase: implications for V(D)J recombination.
Smider V, Rathmell WK, Brown G, Lewis S, Chu G
(1998) Mol Cell Biol 18: 6853-8
MeSH Terms: Animals, DNA, DNA Nucleotidyltransferases, DNA-Activated Protein Kinase, DNA-Binding Proteins, Enzyme Activation, Mice, Mice, SCID, Mutation, Nucleic Acid Conformation, Protein-Serine-Threonine Kinases, Recombination, Genetic, VDJ Recombinases
Show Abstract · Added October 17, 2015
V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.
0 Communities
1 Members
0 Resources
13 MeSH Terms
Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants.
Errami A, Smider V, Rathmell WK, He DM, Hendrickson EA, Zdzienicka MZ, Chu G
(1996) Mol Cell Biol 16: 1519-26
MeSH Terms: Amino Acid Sequence, Animals, Antigens, Nuclear, Autoantigens, Base Sequence, Cell Survival, Cricetinae, Cricetulus, DNA Helicases, DNA Nucleotidyltransferases, DNA-Binding Proteins, Genetic Complementation Test, Ku Autoantigen, Molecular Sequence Data, Mutation, Nuclear Proteins, Radiation Tolerance, Sequence Alignment, VDJ Recombinases
Show Abstract · Added October 17, 2015
X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.
0 Communities
1 Members
0 Resources
19 MeSH Terms