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Pesquisa : D08.811.913.696.445.308 [Categoria DeCS]
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  1 / 5002 MEDLINE  
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[PMID]:28934293
[Au] Autor:Wu J; Liu X; Nayak SG; Pitarresi JR; Cuitiño MC; Yu L; Hildreth BE; Thies KA; Schilling DJ; Fernandez SA; Leone G; Ostrowski MC
[Ad] Endereço:Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America.
[Ti] Título:Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele.
[So] Source:PLoS One;12(9):e0184984, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment.
[Mh] Termos MeSH primário: Transformação Celular Neoplásica/patologia
DNA Nucleotidiltransferases/metabolismo
Modelos Animais de Doenças
Proteínas de Homeodomínio/fisiologia
Neoplasias Pancreáticas/patologia
Transativadores/fisiologia
[Mh] Termos MeSH secundário: Animais
Transformação Celular Neoplásica/genética
Transformação Celular Neoplásica/metabolismo
DNA Nucleotidiltransferases/genética
Progressão da Doença
Feminino
Masculino
Camundongos
Camundongos Endogâmicos C57BL
Neoplasias Pancreáticas/genética
Neoplasias Pancreáticas/metabolismo
Proteínas Proto-Oncogênicas p21(ras)/genética
Transdução de Sinais
Microambiente Tumoral
Proteína Supressora de Tumor p53/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Homeodomain Proteins); 0 (Trans-Activators); 0 (Tumor Suppressor Protein p53); 0 (pancreatic and duodenal homeobox 1 protein); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (FLP recombinase); EC 3.6.5.2 (Kras2 protein, mouse); EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras))
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171017
[Lr] Data última revisão:
171017
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170922
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0184984


  2 / 5002 MEDLINE  
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[PMID]:28734047
[Au] Autor:Baumann M; Gludovacz E; Sealover N; Bahr S; George H; Lin N; Kayser K; Borth N
[Ad] Endereço:Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
[Ti] Título:Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange.
[So] Source:Biotechnol Bioeng;114(11):2616-2627, 2017 Nov.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Site-specific recombinase mediated cassette exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines, this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here, we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers. The alternate start codon reduces the translation initiation efficiency and allows sorting for CHO cells with the highest transcription rates, while RMCE enables the subsequent exchange of the CD4 against the GOI. We have shown that sorted cell pools with the CD4 reporter gene containing the alternate start codon CTG lead to higher GFP signals and higher antibody titers upon RMCE as compared to cell pools containing the ATG start codon of the CD4 reporter. Despite the absence of any subcloning step, the final cell pool contained the CD4 gene in a single genome integration site.
[Mh] Termos MeSH primário: Anticorpos Monoclonais/biossíntese
Anticorpos Monoclonais/genética
Códon de Iniciação/genética
DNA Nucleotidiltransferases/genética
Técnicas de Transferência de Genes
Proteínas Recombinantes/genética
Ativação Transcricional/genética
[Mh] Termos MeSH secundário: Animais
Células CHO
Cricetinae
Cricetulus
Marcação de Genes/métodos
Engenharia de Proteínas/métodos
Transgenes/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibodies, Monoclonal); 0 (Codon, Initiator); 0 (Recombinant Proteins); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (Site-specific recombinase)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171109
[Lr] Data última revisão:
171109
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170723
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26388


  3 / 5002 MEDLINE  
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[PMID]:28660316
[Au] Autor:Jensen IS; Inui K; Drakulic S; Jayaprakash S; Sander B; Golas MM
[Ad] Endereço:Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233, 8000, Aarhus C, Denmark.
[Ti] Título:Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications.
[So] Source:Protein J;36(4):332-342, 2017 Aug.
[Is] ISSN:1875-8355
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3 × FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.
[Mh] Termos MeSH primário: Baculoviridae/genética
Biotecnologia/métodos
DNA Nucleotidiltransferases/genética
DNA/genética
Expressão Gênica
Proteínas de Saccharomyces cerevisiae/genética
[Mh] Termos MeSH secundário: Animais
Baculoviridae/metabolismo
Sítios de Ligação
DNA/metabolismo
DNA Nucleotidiltransferases/metabolismo
Ensaio de Desvio de Mobilidade Eletroforética
Edição de Genes/métodos
Células HEK293
Seres Humanos
Mutação
Oligopeptídeos/genética
Oligopeptídeos/metabolismo
Plasmídeos/química
Plasmídeos/metabolismo
Ligação Proteica
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Saccharomyces cerevisiae/química
Saccharomyces cerevisiae/enzimologia
Proteínas de Saccharomyces cerevisiae/metabolismo
Células Sf9
Spodoptera
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Oligopeptides); 0 (Recombinant Proteins); 0 (Saccharomyces cerevisiae Proteins); 9007-49-2 (DNA); 98849-88-8 (FLAG peptide); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (FLP recombinase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170728
[Lr] Data última revisão:
170728
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170630
[St] Status:MEDLINE
[do] DOI:10.1007/s10930-017-9724-z


  4 / 5002 MEDLINE  
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[PMID]:28472368
[Au] Autor:A Rizvi SM; Prajapati HK; Nag P; Ghosh SK
[Ad] Endereço:Department of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, Maharashtra, India.
[Ti] Título:The 2-µm plasmid encoded protein Raf1 regulates both stability and copy number of the plasmid by blocking the formation of the Rep1-Rep2 repressor complex.
[So] Source:Nucleic Acids Res;45(12):7167-7179, 2017 Jul 07.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The 2-µm plasmid of the budding yeast Saccharomyces cerevisiae achieves a high chromosome-like stability with the help of four plasmid-encoded (Rep1, Rep2, Raf1 and Flp) and several host-encoded proteins. Rep1 and Rep2 and the DNA locus STB form the partitioning system ensuring equal segregation of the plasmid. The Flp recombinase and its target sites FRTs form the amplification system which is responsible for the steady state plasmid copy number. In this work we show that the absence of Raf1 can affect both the plasmid stability and the steady sate copy number. We also show that the Rep proteins do bind to the promoter regions of the 2-µm encoded genes, as predicted by earlier models and Raf1 indeed blocks the formation of the Rep1-Rep2 repressor complex not by blocking the transcription of the REP1 and REP2 genes but by physically associating with the Rep proteins and negating their interactions. This explains the role of Raf1 in both the partitioning and the amplification systems as the Rep1-Rep2 complex is believed to modulate both these systems. Based on this study, we have provided, from a systems biology perspective, a model for the mechanism of the 2-µm plasmid maintenance.
[Mh] Termos MeSH primário: DNA Nucleotidiltransferases/genética
Regulação Fúngica da Expressão Gênica
Plasmídeos/metabolismo
Proteínas Proto-Oncogênicas c-raf/genética
Proteínas de Saccharomyces cerevisiae/genética
Saccharomyces cerevisiae/genética
Transativadores/genética
[Mh] Termos MeSH secundário: Núcleo Celular/genética
Núcleo Celular/metabolismo
Cromossomos/química
Cromossomos/metabolismo
DNA Nucleotidiltransferases/metabolismo
DNA Fúngico/genética
DNA Fúngico/metabolismo
Dosagem de Genes
Loci Gênicos
Plasmídeos/química
Proteínas Proto-Oncogênicas c-raf/metabolismo
Saccharomyces cerevisiae/metabolismo
Proteínas de Saccharomyces cerevisiae/metabolismo
Biologia de Sistemas
Transativadores/metabolismo
Transcrição Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Fungal); 0 (REP1 protein, S cerevisiae); 0 (REP2 protein, S cerevisiae); 0 (Saccharomyces cerevisiae Proteins); 0 (Trans-Activators); EC 2.7.11.1 (Proto-Oncogene Proteins c-raf); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (FLP recombinase)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171017
[Lr] Data última revisão:
171017
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170505
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx316


  5 / 5002 MEDLINE  
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[PMID]:28024983
[Au] Autor:Bowden SD; Palani NP; Libourel IG
[Ad] Endereço:Biotechnology institute, 1500 Gortner Avenue, University of Minnesota, Saint Paul, MN 55108, United States. Electronic address: sbowden@umn.edu.
[Ti] Título:Stringent control of FLP recombinase in Escherichia coli.
[So] Source:J Microbiol Methods;133:52-54, 2017 Feb.
[Is] ISSN:1872-8359
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Site specific recombinases are invaluable tools in molecular biology, and are emerging as powerful recorders of cellular events in synthetic biology. We have developed a stringently controlled FLP recombinase system in Escherichia coli using an arabinose inducible promoter combined with a weak ribosome binding site.
[Mh] Termos MeSH primário: Proteínas de Bactérias/genética
DNA Nucleotidiltransferases/genética
Escherichia coli/enzimologia
Escherichia coli/genética
[Mh] Termos MeSH secundário: Proteínas de Bactérias/metabolismo
Sítios de Ligação
Clonagem Molecular
DNA Helicases/genética
DNA Helicases/metabolismo
DNA Nucleotidiltransferases/metabolismo
Fosfotransferases (Aceptor do Grupo Álcool)/genética
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
Plasmídeos
Regiões Promotoras Genéticas
Recombinação Genética
Transativadores/genética
Transativadores/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Trans-Activators); 0 (replication initiator protein); EC 2.7.1.- (Phosphotransferases (Alcohol Group Acceptor)); EC 2.7.1.16 (ribulokinase); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (FLP recombinase); EC 2.7.7.- (Site-specific recombinase); EC 3.6.4.- (DNA Helicases)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170911
[Lr] Data última revisão:
170911
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161228
[St] Status:MEDLINE


  6 / 5002 MEDLINE  
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[PMID]:27989459
[Au] Autor:Zingg B; Chou XL; Zhang ZG; Mesik L; Liang F; Tao HW; Zhang LI
[Ad] Endereço:Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; Neuroscience Graduate Program, University of Southern California, Los Angeles, CA 90033, USA.
[Ti] Título:AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors.
[So] Source:Neuron;93(1):33-47, 2017 Jan 04.
[Is] ISSN:1097-4199
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:To decipher neural circuits underlying brain functions, viral tracers are widely applied to map input and output connectivity of neuronal populations. Despite the successful application of retrograde transsynaptic viruses for identifying presynaptic neurons of transduced neurons, analogous anterograde transsynaptic tools for tagging postsynaptically targeted neurons remain under development. Here, we discovered that adeno-associated viruses (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties. AAV1-Cre from transduced presynaptic neurons effectively and specifically drives Cre-dependent transgene expression in selected postsynaptic neuronal targets, thus allowing axonal tracing and functional manipulations of the latter input-defined neuronal population. Its application in superior colliculus (SC) reveals that SC neuron subpopulations receiving corticocollicular projections from auditory and visual cortex specifically drive flight and freezing, two different types of defense behavior, respectively. Together with an intersectional approach, AAV-mediated anterograde transsynaptic tagging can categorize neurons by their inputs and molecular identity, and allow forward screening of distinct functional neural pathways embedded in complex brain circuits.
[Mh] Termos MeSH primário: Córtex Auditivo/fisiologia
Dependovirus
Reação de Fuga/fisiologia
Reação de Congelamento Cataléptica/fisiologia
Neurônios/fisiologia
Colículos Superiores/fisiologia
Sinapses/fisiologia
Córtex Visual/fisiologia
[Mh] Termos MeSH secundário: Animais
Córtex Auditivo/citologia
Comportamento Animal/fisiologia
Córtex Cerebral/citologia
Córtex Cerebral/fisiologia
DNA Nucleotidiltransferases
Integrases
Camundongos
Vias Neurais/citologia
Vias Neurais/fisiologia
Colículos Superiores/citologia
Córtex Visual/citologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; VIDEO-AUDIO MEDIA
[Nm] Nome de substância:
EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (FLP recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170803
[Lr] Data última revisão:
170803
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161220
[St] Status:MEDLINE


  7 / 5002 MEDLINE  
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[PMID]:27885986
[Au] Autor:Caspi Y; Dekker C
[Ad] Endereço:Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands.
[Ti] Título:Mapping out Min protein patterns in fully confined fluidic chambers.
[So] Source:Elife;5, 2016 Nov 25.
[Is] ISSN:2050-084X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The bacterial Min protein system provides a major model system for studying reaction-diffusion processes in biology. Here we present the first study of the Min system in fully confined three-dimensional chambers that are lithography-defined, lipid-bilayer coated and isolated through pressure valves. We identify three typical dynamical behaviors that occur dependent on the geometrical chamber parameters: pole-to-pole oscillations, spiral rotations, and traveling waves. We establish the geometrical selection rules and show that, surprisingly, Min-protein spiral rotations govern the larger part of the geometrical phase diagram. Confinement as well as an elevated temperature reduce the characteristic wavelength of the Min patterns, although even for confined chambers with a bacterial-level viscosity, the patterns retain a ~5 times larger wavelength than . Our results provide an essential experimental base for modeling of intracellular Min gradients in bacterial cell division as well as, more generally, for understanding pattern formation in reaction-diffusion systems.
[Mh] Termos MeSH primário: Adenosina Trifosfatases/química
Adenosina Trifosfatases/metabolismo
Proteínas de Ciclo Celular/química
Proteínas de Ciclo Celular/metabolismo
DNA Nucleotidiltransferases/química
DNA Nucleotidiltransferases/metabolismo
Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/metabolismo
Escherichia coli/enzimologia
[Mh] Termos MeSH secundário: Cinética
Conformação Proteica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Cell Cycle Proteins); 0 (Escherichia coli Proteins); 0 (MinE protein, E coli); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (min recombinase); EC 3.6.1.- (Adenosine Triphosphatases); EC 3.6.1.- (MinD protein, E coli)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171002
[Lr] Data última revisão:
171002
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161126
[St] Status:MEDLINE


  8 / 5002 MEDLINE  
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[PMID]:27666751
[Au] Autor:Kato A
[Ad] Endereço:Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University.
[Ti] Título:In vivo cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in Salmonella enterica.
[So] Source:J Gen Appl Microbiol;62(5):225-232, 2016 Nov 25.
[Is] ISSN:1349-8037
[Cp] País de publicação:Japan
[La] Idioma:eng
[Ab] Resumo:Recombineering has been used to facilitate the development of in vivo cloning methods. However, the method relies heavily on PCR, which still generates a much higher error rate than DNA replication in vivo, even when amplifying large DNA inserts. Here, a precise technique is reported in Salmonella enterica that enables the cloning of up to at least 19 kb target chromosomal DNA segments that had been marked by FRTs, which were derived from two consecutive lambda Red-mediated recombination events. P22 phage was utilized to transduce the target DNA segments from donor strains to recipient strains harboring a derivative of bacterial artificial chromosome (BAC) containing a FRT and a plasmid expressing Flp recombinase. This method was successful in cloning a gene cluster responsible for lipopolysaccharide (LPS) modifications that confer polymyxin B resistance and in complementing its mutant. Further optimized procedures should be widely applicable because large insert fragments are precise clones of the wild-type genome.
[Mh] Termos MeSH primário: Cromossomos Artificiais Bacterianos/genética
Clonagem Molecular/métodos
Engenharia Genética/métodos
Recombinação Homóloga
Salmonella typhimurium/genética
Transdução Genética
[Mh] Termos MeSH secundário: Bacteriófago lambda/genética
DNA Nucleotidiltransferases/genética
DNA Nucleotidiltransferases/metabolismo
DNA Bacteriano
Vetores Genéticos
Genoma Bacteriano
Lipopolissacarídeos/genética
Família Multigênica
Plasmídeos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (Lipopolysaccharides); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (FLP recombinase)
[Em] Mês de entrada:1704
[Cu] Atualização por classe:170410
[Lr] Data última revisão:
170410
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160927
[St] Status:MEDLINE


  9 / 5002 MEDLINE  
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[PMID]:27564988
[Au] Autor:Kasai Y; Harayama S
[Ad] Endereço:Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Bunkyo-ku, Tokyo, Japan.
[Ti] Título:Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System.
[So] Source:PLoS One;11(8):e0161733, 2016.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.
[Mh] Termos MeSH primário: Chlamydomonas reinhardtii/genética
Marcadores Genéticos
Organismos Geneticamente Modificados/genética
[Mh] Termos MeSH secundário: Antibacterianos/química
Bleomicina/química
DNA Nucleotidiltransferases
Primers do DNA
Deleção de Genes
Vetores Genéticos
Genoma
Integrases
Plasmídeos/metabolismo
Recombinação Genética
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 0 (DNA Primers); 0 (Genetic Markers); 11056-06-7 (Bleomycin); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (Integrases); EC 2.7.7.- (Site-specific recombinase)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170808
[Lr] Data última revisão:
170808
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160827
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0161733


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[PMID]:27550179
[Au] Autor:Keenholtz RA; Grindley ND; Hatfull GF; Marko JF
[Ad] Endereço:Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.
[Ti] Título:Crossover-site sequence and DNA torsional stress control strand interchanges by the Bxb1 site-specific serine recombinase.
[So] Source:Nucleic Acids Res;44(18):8921-8932, 2016 Oct 14.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:DNA segment exchange by site-specific serine recombinases (SRs) is thought to proceed by rigid-body rotation of the two halves of the synaptic complex, following the cleavages that create the two pairs of exchangeable ends. It remains unresolved how the amount of rotation occurring between cleavage and religation is controlled. We report single-DNA experiments for Bxb1 integrase, a model SR, where dynamics of individual synapses were observed, using relaxation of supercoiling to report on cleavage and rotation events. Relaxation events often consist of multiple rotations, with the number of rotations per relaxation event and rotation velocity sensitive to DNA sequence at the center of the recombination crossover site, torsional stress and salt concentration. Bulk and single-DNA experiments indicate that the thermodynamic stability of the annealed, but cleaved, crossover sites controls ligation efficiency of recombinant and parental synaptic complexes, regulating the number of rotations during a breakage-religation cycle. The outcome is consistent with a 'controlled rotation' model analogous to that observed for type IB topoisomerases, with religation probability varying in accord with DNA base-pairing free energies at the crossover site. Significantly, we find no evidence for a special regulatory mechanism favoring ligation and product release after a single 180° rotation.
[Mh] Termos MeSH primário: DNA Nucleotidiltransferases/metabolismo
DNA/genética
DNA/metabolismo
Recombinação Genética
Proteínas Repressoras/metabolismo
Proteínas Virais/metabolismo
[Mh] Termos MeSH secundário: Sítios de Ligação Microbiológicos
Pareamento de Bases
Clivagem do DNA
Modelos Biológicos
Ligação Proteica
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Repressor Proteins); 0 (Viral Proteins); 0 (gp69 protein, Mycobacteriophage Bxb1); 9007-49-2 (DNA); EC 2.7.7.- (DNA Nucleotidyltransferases); EC 2.7.7.- (Site-specific recombinase)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170606
[Lr] Data última revisão:
170606
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160824
[St] Status:MEDLINE



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