DOCSLIB.ORG

Ability of the Hydrophobic FGF and Basic TAT Peptides To

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Ability of the Hydrophobic FGF and Basic TAT Peptides To Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes Michael Peitz, Kurt Pfannkuche, Klaus Rajewsky*, and Frank Edenhofer† Institute for Genetics, University of Cologne, Weyertal 121, 50931 Cologne, Germany Contributed by Klaus Rajewsky, February 5, 2002 Conditional mutagenesis is a powerful tool to analyze gene func- sive mouse breeding causing the experiments to be time con- tions in mammalian cells. The site-specific recombinase Cre can be suming and costly. The leakiness of the system represents a used to recombine loxP-modified alleles under temporal and spa- critical factor because a Cre recombinase that is undesirably tial control. However, the efficient delivery of biologically active active before induction often leads to unwanted side effects such Cre recombinase to living cells represents a limiting factor. In this as mosaic recombination and͞or selection of recombined or study we compared the potential of a hydrophobic peptide mod- nonrecombined cells both in vivo and in vitro (9, 17, 18). ified from Kaposi fibroblast growth factor with a basic peptide Moreover, the widely used inducers IFN, hydroxy-tamoxifen, derived from HIV-TAT to promote cellular uptake of recombinant and doxycycline are known to display toxic side effects (19, 20) Cre. We present the production and characterization of a Cre and͞or induce also unwanted physiological effects that may protein that enters mammalian cells and subsequently performs interfere with the experimental phenotype of the conditional recombination with high efficiency in a time- and concentration- mutation to be analyzed (21, 22). In cultured cells, Cre-mediated dependent manner. Histidine-tagged Cre recombinase transduced recombination is limited by transfection efficiencies and putative inefficiently unless fused to a nuclear localization signal (NLS). toxicity of the protein (13, 23, 24). Thus, traditional delivery of Fusion of NLS-Cre to the fibroblast growth factor transduction Cre—either by Cre transgenics, viral vectors, or transfection— peptide did not improve the transducibility, whereas fusion with represents a limiting step of conditional mutagenesis employing the TAT peptide significantly enhanced cellular uptake and subse- ͞ quent recombination. More than 95% recombination efficiency in Cre loxP technology. fibroblast cells, as well as murine embryonic stem cells, was Protein transduction is a recently developed method to intro- achieved after His-TAT-NLS-Cre transduction. Efficient recombina- duce biologically active proteins directly into mammalian cells tion could also be obtained in primary splenocytes ex vivo.We with high efficiency (for review, see refs. 25 and 26). Recombi- expect that application of His-TAT-NLS-Cre, which can be produced nant technology has been used to modify biophysical properties readily in large quantities from a bacterial source, will expand our of proteins, particularly with respect to their cell permeability, by abilities to manipulate mammalian genomes. employing so-called protein transduction domains (PTDs) (27– 29). It has been demonstrated that a basic 11-aa peptide, derived from HIV-TAT, renders ␤-galactosidase (␤-gal) into a cell- onditional mutagenesis in mammalian cells has become an GENETICS permeable form. ␤-gal activity can be detected in organs such as important means for the analysis of gene function in vivo (for C liver, kidney, lung, brain, and spleen of mice after i.p. injection review, see refs. 1–3). Site-specific recombinases such as the ␤ bacteriophage P1 recombinase Cre have been used to gain of TAT- -gal (28). Besides the basic TAT peptide, other pep- control over the mutation in a spatial (4–6) and͞or temporal tides or protein (fragments) also have been reported to enhance manner (7, 8). An increasing number of studies have demon- cellular uptake of proteins, such as a hydrophobic peptide strated the efficacy of Cre-mediated conditional mutagenesis in modified from Kaposi fibroblast growth factor (FGF) (29, 30). mice and cell lines (9–12). Usually a mouse or cell line is Recently it has been reported that a fusion protein of a nuclear generated, in which an essential part of the gene of interest is localization signal (NLS), Cre recombinase, and a FGF peptide flanked by two loxP sites. The loxP sites represent Cre recom- displays cell permeability. After direct application of the recom- binase recognition sites and can be used to delete the respective binant protein to cultured cells ofaTlymphocyte line containing gene segment upon Cre recombination, resulting in a condi- a loxP-modified substrate, Cre-mediated recombination was tioned inactivation or mutation of the gene of interest. To gain observed in Ϸ80% of the cells (31). However, it remained temporal control over this mutation event two different ap- unclear from this study whether the FGF peptide is essential for proaches have been applied: (i) Cre is delivered into cultured transduction or even contributes significantly to the cellular cells either by transfection (13) or adenoviral infection (14, 15); uptake of Cre. (ii) Cre recombinase activity is induced by application of an exogenous inducer. Induction can be carried out either at the transcriptional level [e.g., Mx-Cre (7) or tetracycline-controlled Abbreviations: aa, amino acid(s); ␤-gal, ␤-galactosidase; ES, embryonic stem cell(s); FGF, fibroblast growth factor; NLS, nuclear localization signal; PTD, protein transduction Cre expression (16)] or posttranslational level employing fusion domain. proteins of Cre with mutated ligand-binding domains of steroid *Present address: Center for Blood Research, Harvard Medical School, 200 Longwood receptors (8). Although a number of studies demonstrated the Avenue, Boston, MA 02115. efficacy of Cre-mediated inducible mutagenesis, still the system †To whom reprint requests should be addressed. E-mail: [email protected]. could profit substantially from technical advance concerning The publication costs of this article were defrayed in part by page charge payment. This three major aspects: leakiness of the system before induction, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. efficiency of induced recombination, and requirement of exten- §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.032068699 PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 ͉ 4489–4494 Downloaded by guest on October 2, 2021 Our study was aimed at evaluating the actual potency of two Overexpression was induced by adding IPTG to a final concen- prominent PTDs, namely FGF and TAT peptides, to promote tration of 0.5 mM and an additional incubation of 3–4hat37°C. the translocation of biologically active Cre across the plasma Cells were harvested by centrifugation and frozen in dry ice͞ membrane of mammalian cells. We generated expression vectors ethanol and stored at Ϫ20°C. Cell pellets were thawed by ͞ encoding Cre recombinase fused to FGF and TAT peptides, resuspending in lysis buffer [100 mM NaH2PO4 10 mM Tris (pH respectively. The potentials of the recombinant proteins to 8.0)͞300 mM NaCl͞10 mM imidazole] at 5 ml per gram wet transduce and subsequently recombine loxP-flanked targets in weight. Cleared lysate was obtained after incubation with 2 mammalian cells were compared side by side with Cre lacking mg͞ml lysozyme (Sigma) and benzonase (Novagen) and centrif- any particular PTD. ugation for 25 min at 30,000 ϫ g at 4°C. One milliliter of 50% Ni-NTA slurry (Qiagen) was added to 5 ml of cleared lysate and Materials and Methods mixed gently by shaking at 4°C for 1 h. The slurry was packed into Plasmid Constructions. A TAT-encoding fragment was generated a column and washed with 10 bed volumes [100 mM ͞ ͞ ͞ by PCR, using primers NPTatU and NeuUS. This fragment was NaH2PO4 10 mM Tris (pH 8.0) 300 mM NaCl 20 mM imida- ͞ cloned into the pTriEx-1 vector (Novagen) via NcoI and XhoI zole]. Recombinant protein was eluted [100 mM NaH2PO4 10 restriction sites, resulting in pTriEx-TAT-U-H (F.E. and K.R., mM Tris (pH 8.0)͞300 mM NaCl͞250 mM imidazole] and either unpublished data). This vector encodes, in addition to TAT, a dialyzed against appropriate media (see below) for immediate second protein fragment designated as ‘‘U,’’ which was not used use or frozen at Ϫ20°C for storage up to 8 weeks without in this study. The corresponding DNA fragment was deleted by significant loss of activity. Protein concentrations were measured digestion with SpeI and subsequent religation, resulting in using Bradford reagent (Sigma) and͞or Warburg method. Cre pTriEx-TAT-H. The TAT-encoding fragment was removed, activities were determined by a cell-free assay as described (31, resulting in pTriEx-H by PstI digestion. To obtain pTriEx-CH,a 33), except for our use of 250 ng of substrate DNA (33). For Cre-encoding PCR product was generated from the template quantification reactions were transformed into E. coli and pPGK-Cre-bpA (32) by using primers 5H3cre and 3X1cre. This colonies counted. A commercially available Cre protein (New fragment was subsequently cloned into the pTriEx-H by using England Biolabs) was used as a standard. HindIII and XhoI sites. To generate pTriEx-NCH, a NLS-Cre- encoding fragment was amplified using primers 5H3NLScre and SDS͞PAGE and Immunoblot Analysis. Samples were separated by 3X1cre and cloned as above. To construct pTriEx-FNCH, an- SDS͞PAGE (10%), transferred to nitrocellulose membranes nealed oligonucleotides 5PstFGF and 3PstFGF were cloned into (Amersham Pharmacia), and probed with anti-Cre (Novagen) or the PstI restriction site of pTriEx-NCH. To generate pTriEx- anti-Penta-His (Qiagen) antiserum. Anti-Cre was incubated HTNC, a NLS-Cre-STOP encoding PCR product was generated 1:5,000 in PBS containing 5% dry milk, and anti-Penta-His was using primers 5H3NLScre and 3creStopXho and subsequently used 1:2,000 in PBS containing 3% BSA. Blocking was carried cloned into pTriEx-TAT-H via HindIII and XhoI restriction out in corresponding buffers without antibody for1hatroom sites; the His tag was introduced by cloning annealed oligonu- temperature.
Load more

Recommended publications
  • An Overview of Tyrosine Site-Specific Recombination: from an Flp Perspective
    An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective MAKKUNI JAYARAM,1 CHIEN-HUI MA,1 AASHIQ H KACHROO,1 PAUL A ROWLEY,1 PIOTR GUGA,2 HSUI-FANG FAN,3 and YURI VOZIYANOV4 1Department of Molecular Biosciences, UT Austin, Austin, TX 78712; 2Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bio-organic Chemistry, Lodz, Poland; 3Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan; 4School of Biosciences, Louisiana Tech University, Ruston, LA 71272 ABSTRACT Tyrosine site-specific recombinases (YRs) are prokaryotes. They were thought to be nearly absent widely distributed among prokaryotes and their viruses, and among eukaryotes, the budding yeast lineage (Saccharo- fi were thought to be con ned to the budding yeast lineage mycetaceae) being an exception in that a subset of its among eukaryotes. However, YR-harboring retrotransposons members houses nuclear plasmids that code for YRs (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize (1, 2). However, YR-harboring DIRS and PAT families a common chemical mechanism, analogous to that of type IB of retrotransposons and presumed DNA transposons topoisomerases, to bring about a plethora of genetic classified as Cryptons have now been identified in a rearrangements with important physiological consequences large number of eukaryotes (3, 4). The presence of in their respective biological contexts. A subset of the tyrosine functional YRs encoded in Archaeal genomes has been recombinases has provided model systems for analyzing the established by a combination of comparative genomics chemical mechanisms and conformational features of the and modeling complemented by biochemical and struc- recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical tural analyses (5, 6).
    [Show full text]
  • Chimeras of the Flp and Cre Recombinases: Tests of the Mode of Cleavage by Flp and Cre A
    doi:10.1006/jmbi.2000.3967 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 302, 27±48 Chimeras of the Flp and Cre Recombinases: Tests of the Mode of Cleavage by Flp and Cre A. C. Shaikh and Paul D. Sadowski* Department of Molecular and The Flp and Cre recombinases are members of the integrase family of Medical Genetics, University of tyrosine recombinases. Each protein consists of a 13 kDa NH2-terminal Toronto, Toronto, M5S 1A8 domain and a larger COOH-terminal domain that contains the active site Canada of the enzyme. The COOH-terminal domain also contains the major determinants for the binding speci®city of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attach- ment of a conserved nucleophilic tyrosine residue to the 30-phosphate group at the sites of cleavage. In order to gain further insights into the determinants of the binding speci®city and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH2-terminal domain of Flp to the COOH-terminal domain of Cre (``Fre'') and the NH2-terminal domain of Cre to the COOH-terminal domain of Flp (``Clp''). These chimeras have novel binding speci®cities in that they bind strongly to hybrid sites con- taining elements from both the Flp and Cre DNA targets but poorly to the native target sites. In this study we have taken advantage of the unique binding speci®ci- ties of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp.
    [Show full text]
  • Quick & Easy Conditional Knockout Kit (Loxp/Cre)
    Technical Protocol Cat. No. K005 Quick & Easy Conditional Knockout Kit (loxP/Cre) By Red®/ET® Recombination Version 1.3 (May 2014) CONTENTS 1 Conditional Knockout Kit (loxP/Cre) ........................................................................................... 3 2 Experimental Outline .................................................................................................................... 6 3 How Red/ET Recombination works ............................................................................................ 8 4 Oligonucleotide Design for Red/ET Recombination ............................................................... 10 5 Media for Antibiotic Selection ................................................................................................... 12 6 Technical protocol ...................................................................................................................... 13 6.1 Generation of a functional cassette flanked by homology arms .......................................... 13 6.2 Transformation with Red/ET expression plasmid pRedET .................................................. 14 6.3 Insertion of the loxP flanked PGK-gb2-neo cassette into a plasmid .................................... 16 6.4 Verification of successfully modified plasmid by restriction analysis ................................... 19 6.5 Deletion of the kanamycin/neomycin selection marker by Cre expression ......................... 21 6.6 Verification of successfully modified plasmid by restriction analysis
    [Show full text]
  • TRF2 Deletion
    LETTERS DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion Giulia B. Celli1,2 and Titia de Lange1,3 Telomere attrition and other forms of telomere damage can has been implicated in the Mec1-dependent response to double-strand activate the ATM kinase pathway. What generates the DNA breaks (DSBs)8. However, in mammalian cells it is not known whether damage signal at mammalian chromosome ends or at other or how DSBs are processed, and what the signal for ATM activation is double-strand breaks is not known. Telomere dysfunction remains a subject for debate9. Telomeres represent a unique opportunity is often accompanied by disappearance of the 3´ telomeric to study these issues because they provide molecularly marked sites that overhang1,2, raising the possibility that DNA degradation could can activate the canonical DNA damage response. generate the structure that signals2. Here we address these To study the DNA damage response at telomeres, we targeted the issues by studying telomere structure after conditional deletion mouse TRF2 gene on chromosome 8 (Terf2; Mouse Genome Informatics of mouse TRF2, the protective factor at telomeres. Upon (MGI) number 1195972; referred to hereafter as TRF2 for clarity). The removal of TRF2 from TRF2F/– p53–/– mouse embryo fibroblasts, TRF2 locus was modified such that exon 1 (containing the translation a telomere damage response is observed at most chromosome initiation site) and exon 2 are flanked by loxP sites, allowing inacti- ends. As expected, the telomeres lose the 3´ overhang and are vation of the TRF2 gene with Cre recombinase (Fig.
    [Show full text]
  • Different Requirements of Functional Telomeres in Neural Stem Cells and Terminally Differentiated Neurons
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Different requirements of functional telomeres in neural stem cells and terminally differentiated neurons Anastasia Lobanova,1 Robert She,1 Simon Pieraut,2,3 Charlie Clapp,1,4 Anton Maximov,2 and Eros Lazzerini Denchi1 1Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037, USA; 2Department of Neuroscience, The Scripps Research Institute, La Jolla, California 92037, USA Telomeres have been studied extensively in peripheral tissues, but their relevance in the nervous system remains poorly understood. Here, we examine the roles of telomeres at distinct stages of murine brain development by using lineage-specific genetic ablation of TRF2, an essential component of the shelterin complex that protects chromo- some ends from the DNA damage response machinery. We found that functional telomeres are required for em- bryonic and adult neurogenesis, but their uncapping has surprisingly no detectable consequences on terminally differentiated neurons. Conditional knockout of TRF2 in post-mitotic immature neurons had virtually no detectable effect on circuit assembly, neuronal gene expression, and the behavior of adult animals despite triggering massive end-to-end chromosome fusions across the brain. These results suggest that telomeres are dispensable in terminally differentiated neurons and provide mechanistic insight into cognitive abnormalities associated with aberrant telo- mere length in humans. [Keywords: dentate gyrus; neurons; telomeres] Supplemental material is available for this article. Received January 6, 2017; revised version accepted March 16, 2017. Telomeres are nucleoprotein structures at chromosome Polvi et al. 2012; Savage 2014). While characterized by cel- ends.
    [Show full text]
  • A Novel Mutant of Cre–PR Fusion Protein Exhibits Enhanced Sensitivity and an Expanded Range of Inducibility Frank T
    © 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 10 e47 New variants of inducible Cre recombinase: a novel mutant of Cre–PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility Frank T. Wunderlich, Hendrik Wildner, Klaus Rajewsky and Frank Edenhofer* Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany Received January 11, 2001; Revised and Accepted March 21, 2001 ABSTRACT applying high doses of inducer (6,7). Secondly, Cre–LBD We have developed a novel inducible Cre mutant with fusion proteins exhibit unwanted residual recombinase activity in the absence of inducer in vivo (7) and, particularly, in vitro enhanced recombinase activity to mediate genetic (4,10,11). Leakiness of the system is a major drawback, since switching events. The protein, designated Cre*PR, is every recombination event is irreversible and therefore even low composed of a new Cre mutant at the N-terminus background recombinase activity may result in high accumulation followed by the ligand-binding domain (LBD) of the of recombined target genes. Unambiguous switching of genes progesterone receptor (PR). The response to low using the Cre–LBD system is hindered by ligand-independent doses of inducer is significantly enhanced by elon- recombinase activity (4,10). It has been suggested that basal gating the C-terminus of the PR LBD from amino acid activity may result from proteolysis of Cre–LBD fusion 891 to 914. The mutant Cre lacks the first 18 amino proteins (4) or from cryptic splicing of the corresponding acids and contains a Val→Ala substitution at position mRNA.
    [Show full text]
  • Site-Specific Recombination with Inverted Target Sites: a Cautionary Tale of Dicentric And
    Genetics: Early Online, published on June 25, 2020 as 10.1534/genetics.120.303394 Site-specific recombination with inverted target sites: a cautionary tale of dicentric and acentric chromosomes Simon W. A. Titen1, Makenna T. B. Johnson2, Mario Capecchi1, Kent G. Golic2* 1 Eccles Inst. of Human Genetics University of Utah School of Medicine 15 North 2030 East, Rm. 5440 Salt Lake City, UT, 84112 USA 2 School of Biological Sciences University of Utah 257 South 1400 East Salt Lake City, UT, 84112 USA * corresponding author (801)581-8726 [email protected] 1 Copyright 2020. ABSTRACT Site-specific recombinases are widely used tools for analysis of genetics, development and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the consequence of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.
    [Show full text]
  • Site-Specific DNA Recombinationin Mammalian Cells by the Cre
    Proc. Nail. Acad. Sci. USA Vol. 85, pp. 5166-5170, July 1988 Genetics Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1 (genome rearrangement/herpesviruses/Cre-lox/metalothionein) BRIAN SAUER AND NANCY HENDERSON E. I. du Pont de Nemours and Company, Inc., Central Research and Development Department, Experimental Station, Wilmington, DE 19898 Communicated by Robert A. Weinberg, March 28, 1988 ABSTRACT The Cre protein encoded by the coliphage P1 give inversion of the intervening DNA segment. Both intra- is a 38-kDa protein that efficiently promotes both intra- and and intermolecular recombination is catalyzed by Cre with intermolecular synapsis and recombination of DNA both in either supercoiled or linear DNA. Recently we have shown Escherichia coli and in vitro. Recombination occurs at a specific that the Cre-lox system can function efficiently in yeast to site, called lox, and does not require any other protein factors. cause recombination on chromosomes present in the nucleus The Cre protein is shown here also to be able to cause synapsis (5). We now demonstrate that this site-specific recombination of DNA and site-specific recombination in a mammalian cell system also functions in a mammalian cell. line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallo- MATERIALS AND METHODS thionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. Plasmids. The Cre expression plasmid pBS31 and its One such substrate is a circular plasmid with two directly parent, pBMTx (obtained from G.
    [Show full text]
  • Recent Advances in Gene Mutagenesis by Site-Directed Recombination
    Recent advances in gene mutagenesis by site-directed recombination. J D Marth J Clin Invest. 1996;97(9):1999-2002. https://doi.org/10.1172/JCI118634. Perspective Find the latest version: https://jci.me/118634/pdf Perspectives Series: Molecular Medicine in Genetically Engineered Animals Recent Advances in Gene Mutagenesis by Site-directed Recombination Jamey D. Marth Howard Hughes Medical Institute, Department of Medicine and Division of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093 Transgenic experimentation yields insights that could not be lack of cell or tissue development may result from ES cell perceived otherwise among populations of mammalian organ- clonal variation, use of multiple ES clones and complementa- isms (reviewed in references 1 and 2). However, information tion by gene transfer allow for controlled studies. Some con- gained may, on occasion, be somewhat limited should germ- siderations remain the inability to direct genetic variation to line gene dysfunction be deleterious in embryonic develop- multiple, experimentally defined cell lineages and the poten- ment, thereby precluding analyses in various somatic compart- tially unique nature of each chimeric mouse generated. ments of the adult organism. The production of chimeric mice Since inception, gene transfer experimentation has been bearing genetic mutations specifically in various somatic cells generally limited to irreversible modifications of chromosomal allows the study of gene function in physiologic contexts that DNA. Isolation of cells having undergone site-specific ex- would otherwise be unavailable or lethal. One elegant cell change or deletion of DNA sequence has only recently been type-restricted chimeric approach has been the use of the developed using novel screening strategies for often low fre- RAG 2-null embryos as recipients in gene-targeted embryonic quency events.
    [Show full text]
  • Specific Cre/Lox Recombination in the Mouse Proximal Tubule
    J Am Soc Nephrol 15: 2050–2056, 2004 Specific Cre/Lox Recombination in the Mouse Proximal Tubule ISABELLE RUBERA, CHANTAL POUJEOL, GUILLAUME BERTIN, LILIA HASSEINE, LAURENT COUNILLON, PHILIPPE POUJEOL, and MICHEL TAUC UMR-CNRS 6548, University of Nice-Sophia Antipolis, Parc Valrose, Nice, France Abstract. The present work reports for the first time the con- tion of Cre but not for the other tissues analyzed. Within the struction of a transgenic mouse strain with specific expression kidney, Cre transcripts were demonstrated to be restricted to of Cre recombinase in the kidney proximal tubule. A Cre/loxP the proximal tubule only. iL1-sglt2-Cre mice were bred with strategy was developed using sglt2 promoter to drive Cre ROSA26-LacZ reporter mice that contained a loxP-flanked recombinase expression in transgenic mice. The mouse sglt2 5' stop sequence upstream of the LacZ gene. X-gal staining and region consisting of the first exon, the first intron, and part of immunohistochemistry using specific antibodies (anti-megalin, the second exon was cloned upstream of a nucleotide sequence anti–Tamm-Horsfall, anti–NaCl co-transporter, and anti–aqua- encoding the Cre recombinase. Transgenic mice were gener- porin 2) revealed that sglt2 drives Cre functional expression ated by pronuclear injection, and tissue specificity of Cre specifically in proximal tubules. The iL1-sglt2-Cre mouse expression was analyzed using reverse transcription–PCR. The therefore represents a powerful tool for Cre-LoxP–mediated iL1-sglt2-Cre mouse line scored positive for kidney transcrip- conditional expression in the renal proximal tubule. The kidney is the organ that maintains body fluid homeostasis.
    [Show full text]
  • The Cre Recombinase Gene Y
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3932-3936, April 1996 Neurobiology Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene Y. WANG*, L. A. KRUSHEL*t, AND G. M. EDELMAN*t *Department of Neurobiology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037; and tThe Neurosciences Institute, 10640 John Jay Hopkins Road, San Diego, CA 92121 Contributed by G. M. Edelman, December 26, 1995 ABSTRACT Conditional gene expression and gene dele- sequences, each of which is 34 bp in length (7, 8). The tion are important experimental approaches for examining intramolecular recombination of two loxP sites (oriented ei- the functions of particular gene products in development and ther head-to-head or head-to-tail) results in an inversion or disease. The cre-loxP system from bacteriophage P1 has been deletion of the intervening DNA sequences, and an intermo- used in transgenic animals to induce site-specific DNA re- lecular recombination causes integration or reciprocal trans- combination leading to gene activation or deletion. To regulate location at the loxP site. The cre-loxP system has been tested the recombination in a spatiotemporally controlled manner, in various eukaryotic organisms and shown to function effi- we constructed a recombinant adenoviral vector, Adv/cre, that ciently in mammalian cells (9-13). It has been used in trans- contained the cre recombinase gene under regulation of the genic animals to achieve conditional gene activation or dele- herpes simplex virus thymidine kinase promoter. The efficacy tion (knockout) of targeted genes in specific tissues (5, 6, 14).
    [Show full text]
  • Endothelial Senescence-Associated Secretory Phenotype (SASP) Is Regulated by Makorin-1 Ubiquitin E3 Ligase
    Metabolism Clinical and Experimental 100 (2019) 153962 Contents lists available at ScienceDirect Metabolism Clinical and Experimental journal homepage: www.metabolismjournal.com Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase Sivareddy Kotla a,⁎, Nhat-Tu Le b,HangThiVua, Kyung Ae Ko a, Young Jin Gi a,TamlynN.Thomasa, Carolyn Giancursio a, Aldos J. Lusis c,JohnP.Cookeb, Keigi Fujiwara a, Jun-ichi Abe a,⁎ a Department of Cardiology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA b Center for Cardiovascular Regeneration, Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX, USA c Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA article info abstract Article history: Background: Disturbed flow (d-flow)–induced senescence and activation of endothelial cells (ECs) have been Received 5 March 2019 suggested to have critical roles in promoting atherosclerosis. Telomeric repeat-binding factor 2 (TERF2)- Accepted 21 August 2019 interacting protein (TERF2IP), a member of the shelterin complex at the telomere, regulates the senescence- associated secretory phenotype (SASP), in which EC activation and senescence are engendered simultaneously Keywords: by p90RSK-induced phosphorylation of TERF2IP S205 and subsequent nuclear export of the TERF2IP-TERF2 com- Telomeric repeat binding factor 2-interacting plex. In this study, we investigated TERF2IP-dependent gene expression and its role in regulating d-flow-induced protein (TERF2IP) SASP. Senescence-associated secretory phenotype (SASP) Methods: A principal component analysis and hierarchical clustering were used to identify genes whose expres- p90RSK sion is regulated by TERF2IP in ECs under d-flow conditions.
    [Show full text]