RESUMO
The erythropoietin receptor (EpoR) has traditionally been thought of as an erythroid-specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In this study, we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice, in which a very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes, and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further shown by the EpoR-tdTomato-Cre-mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles for EPO/EpoR in hematopoiesis that warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.
Assuntos
Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Receptores da Eritropoetina/genética , Animais , Células-Tronco Hematopoéticas/citologia , Humanos , Integrases/análise , Integrases/genética , Substâncias Luminescentes/análise , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Macrófagos/citologia , Camundongos , Receptores da Eritropoetina/análise , Proteína Vermelha FluorescenteRESUMO
Transgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. The investigation of interneurons in particular has benefitted from the ability to genetically target specific cell types. However, the specificity of some Cre driver lines has been called into question. Here, we show that nonspecific expression in a subset of hippocampal neurons can have substantial nonspecific functional effects in a somatostatin-Cre (SST-Cre) mouse line. Nonspecific targeting of CA3 pyramidal cells caused large optogenetically evoked excitatory currents in remote brain regions. Similar, but less severe patterns of nonspecific expression were observed in a widely used SST-IRES-Cre line, when crossed with a reporter mouse line. Viral transduction on the other hand yielded more specific expression but still resulted in nonspecific expression in a minority of pyramidal layer cells. These results suggest that a careful analysis of specificity is mandatory before the use of Cre driver lines for opto- or chemogenetic manipulation approaches.
Assuntos
Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/metabolismo , Integrases/biossíntese , Interneurônios/metabolismo , Optogenética/métodos , Somatostatina/biossíntese , Animais , Região CA3 Hipocampal/química , Expressão Gênica , Integrases/análise , Integrases/genética , Interneurônios/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Somatostatina/análise , Somatostatina/genéticaRESUMO
The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.SIGNIFICANCE STATEMENT The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.
Assuntos
Perfilação da Expressão Gênica/métodos , Integrases/análise , Mesencéfalo/química , Neurônios/química , Pseudorraiva , Recompensa , Animais , Feminino , Integrases/metabolismo , Masculino , Mesencéfalo/anatomia & histologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Pseudorraiva/metabolismoRESUMO
Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.
Assuntos
Antivirais/farmacologia , Retrovirus Endógenos/enzimologia , Retrovirus Endógenos/fisiologia , Integrases/análise , Raltegravir Potássico/farmacologia , Integração Viral/efeitos dos fármacos , Animais , Antivirais/química , Cristalografia por Raios X , Retrovirus Endógenos/efeitos dos fármacos , Concentração Inibidora 50 , Integrases/química , Ligação Proteica , Conformação Proteica , Raltegravir Potássico/química , SuínosRESUMO
There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP(+) cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.
Assuntos
Proteínas de Fluorescência Verde/análise , Integrases/análise , Neurônios/química , Optogenética/métodos , Retina/química , Retina/citologia , Animais , Feminino , Células HEK293 , Humanos , Integrases/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Retina/metabolismoRESUMO
Class 1 integrons are mobile gene elements (MGEs) containing qacEΔ1 that are resistant to quaternary ammonium compound (QAC) disinfectants. This study compared the abundances of class 1 integrons and antiseptic resistance genes in municipal (M) and swine slaughterhouse (S) wastewater treatment plants (WWTPs) and investigated the presence of class 1 integrons and antiseptic resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) isolated from wastewater samples. The abundances of intI1 and qacEΔ1 genes in 96 wastewater samples were quantified using real-time quantitative polymerase chain reaction (real-time qPCR), and 113 MRSA isolates recovered from the wastewater samples were detected class 1 integrons and linked antiseptic resistance genes (qacEΔ1), and minimum inhibitory concentrations (MICs) for QAC antiseptics. The intI1 and qacEΔ1 genes were detected in all the wastewater samples, and they were more abundant in S-WWTP samples than in M-WWTP samples. A higher percentage of MRSA isolates carried qacEΔ1 in MRSA from swine wastewater samples (62.8%) than in municipal MRSA (3.7%). All the MRSA isolates showed high MICs for antiseptic agents. This study provides important evidence regarding the abundances of intI1 and qacEΔ1 genes in municipal and swine slaughterhouse wastewater, and antiseptic-resistant MRSA strains were detected in swine slaughterhouse wastewater.
Assuntos
Integrons , Staphylococcus aureus Resistente à Meticilina/genética , Águas Residuárias/química , Matadouros , Animais , Anti-Infecciosos Locais , Proteínas de Bactérias/genética , Desinfetantes , Integrases/análise , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Águas Residuárias/microbiologiaRESUMO
Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1) in raw sewage (RS) and activated sludge (AS). The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS) and 92% (RS) of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS) as well as for Enhydrobacter (RS). The activated sludge process decreased (55%) the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.
Assuntos
Bactérias , Integrases/genética , Microbiota/genética , Esgotos/microbiologia , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Brasil , Variação Genética , Humanos , Integrases/análise , Clima Tropical , Águas Residuárias/análise , Águas Residuárias/microbiologia , Purificação da ÁguaRESUMO
We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.
Assuntos
Células da Medula Óssea/citologia , Medula Óssea/ultraestrutura , Integrases/análise , Células-Tronco/citologia , Células Estromais/citologia , Fatores de Transcrição/análise , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Camundongos , Fator de Transcrição Sp7 , Coloração e Rotulagem/métodosRESUMO
Helper-dependent adenoviral vectors (HD Ad) hold extreme promise for gene therapy of human diseases. All viral genes are deleted in HD Ad vectors, and therefore, the presence of a helper virus is required for their production. Current methods to minimize helper contamination in large-scale preparations rely on the use of the Cre/loxP system. The inclusion of loxP sites flanking the packaging signal results in its excision in the presence of Cre recombinase, preventing helper genome encapsidation. It is well established that the level of Cre recombinase activity is important in determining the degree of helper contamination. However, there is little information on other mechanisms that could also play an important role. We have generated several HD Ad vectors containing a rapalog-inducible system to regulate transgene expression, or LacZ under the control of the elongation factor 1 α promoter. Large-scale production of these vectors resulted in abundant helper contamination. Viral DNA analysis revealed the presence of rearrangements between vector and helper genomes. The rearrangements involved a helper DNA molecule with a fragment of the left arm of the HD Ad vector, including its ITR, packaging signal, and some stuffer sequence. Overall, our data suggest that helper DNA molecules that accumulate after Cre recombinase activity are prone to rearrangements, resulting in helper genomes that have incorporated a packaging signal from the vector. Helper particles with rearranged genomes have a growth advantage. This study identifies a novel mechanism leading to helper contamination during helper-dependent adenoviral vector production.
Assuntos
Adenoviridae/genética , DNA Viral/isolamento & purificação , Rearranjo Gênico , Vetores Genéticos , Vírus Auxiliares/genética , Western Blotting , DNA Viral/genética , Regulação da Expressão Gênica , Terapia Genética , Células HEK293 , Humanos , Integrases/análise , Integrases/metabolismo , Óperon Lac , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transgenes , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The objective of this research was to investigate the ability of anaerobic and aerobic digesters to reduce the quantity of antibiotic resistant bacteria in wastewater solids. Lab-scale digesters were operated at different temperatures (22 °C, 37 °C, 46 °C, and 55 °C) under both anaerobic and aerobic conditions and fed wastewater solids collected from a full-scale treatment facility. Quantitative PCR was used to track five genes encoding tetracycline resistance (tet(A), tet(L), tet(O), tet(W), and tet(X)) and the gene encoding the integrase (intI1) of class 1 integrons. Statistically significant reductions in the quantities of these genes occurred in the anaerobic reactors at 37 °C, 46 °C, and 55 °C, with the removal rates and removal efficiencies increasing as a function of temperature. The aerobic digesters, in contrast, were generally incapable of significantly decreasing gene quantities, although these digesters were operated at much shorter mean hydraulic residence times. This research suggests that high temperature anaerobic digestion of wastewater solids would be a suitable technology for eliminating various antibiotic resistance genes, an emerging pollutant of concern.
Assuntos
Temperatura Alta , Integrases/genética , Resistência a Tetraciclina/genética , Eliminação de Resíduos Líquidos/métodos , Aerobiose , Anaerobiose , Bactérias Aeróbias/genética , Bactérias Anaeróbias/genética , Reatores Biológicos/microbiologia , Cidades , Genes Bacterianos , Integrases/análise , Integrons/genéticaRESUMO
Pancreatic beta-cells are critical regulators of glucose homeostasis, and they vary dramatically in their glucose stimulated metabolic response and levels of insulin secretion. It is unclear whether these parameters are influenced by the developmental origin of individual beta-cells. Using HOTcre, a Cre-based genetic switch that uses heat-induction to precisely control the temporal expression of transgenes, we labeled two populations of beta-cells within the developing zebrafish pancreas. These populations originate in distinct pancreatic buds and exhibit gene expression profiles suggesting distinct functions during development. We find that the dorsal bud derived beta-cells are quiescent and exhibit a marked decrease in insulin expression postembryonically. In contrast, ventral bud derived beta-cells proliferate actively, and maintain high levels of insulin expression compared with dorsal bud derived beta-cells. Therapeutic strategies to regulate beta-cell proliferation and function are required to cure pathological states that result from excessive beta-cell proliferation (e.g., insulinoma) or insufficient beta-cell mass (e.g., diabetes mellitus). Our data reveal the existence of distinct populations of beta-cells in vivo and should help develop better strategies to regulate beta-cell differentiation and proliferation.
Assuntos
Proliferação de Células , Genes Reporter , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Integrases/análise , Interfase , Peixe-Zebra/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Insulina/metabolismo , Integrases/genética , Integrases/metabolismo , Técnicas de Sonda Molecular , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The pig is considered to be the most suitable nonhuman source of organs for xenotransplantation and is widely used as a model of human disease. The Cre-LoxP system provides a powerful means of cell- or tissue-specific deletion of a targeted gene in cells or tissues of interest. Pigs expressing Cre recombinase have a profound impact on the study of gene function and the generation of animal models of human diseases. To monitor Cre recombinase expression in vivo, it is important to create reporter strains. As a first step in the production of such transgenic pigs, we generated porcine fetal fibroblast cell lines conditionally expressing the gene for enhanced green fluorescent protein (EGFP). The EGFP gene is expressed only after Cre-mediated excision of LoxP-flanked stop sequences. These fetal fibroblast cell lines will be of great value for constructing reporter transgenic pigs.
Assuntos
Linhagem Celular , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Integrases/análise , Sus scrofa/embriologia , Transgenes , Animais , Animais Geneticamente Modificados , Feto/citologia , Citometria de Fluxo , Genes Sintéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Microscopia Confocal , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa/genética , TransfecçãoRESUMO
The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type- specific promoter. We used 7 kb of the ATP6V1B1 5' untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.
Assuntos
Integrases/biossíntese , Túbulos Renais Coletores/enzimologia , Rim/citologia , Regiões Promotoras Genéticas/fisiologia , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Feminino , Genitália Masculina , Humanos , Integrases/análise , Rim/enzimologia , Proteínas Luminescentes , Masculino , Camundongos , Camundongos Transgênicos , ATPases Vacuolares Próton-Translocadoras/análiseRESUMO
During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.
Assuntos
Integrases/genética , Camundongos Transgênicos/embriologia , Camundongos Transgênicos/genética , Notocorda/metabolismo , Organizadores Embrionários/metabolismo , Animais , Fator 3-beta Nuclear de Hepatócito/genética , Integrases/análise , Integrases/metabolismo , Camundongos , Mutagênese , Proteína Nodal , Elementos Reguladores de Transcrição , Fator de Crescimento Transformador beta/genéticaRESUMO
We have knocked-in Cre-IRES-EGFP in the Foxb1 locus by homologous recombination in embryonic stem cells. We removed the PGK-neo cassette (which was flanked by FRT sequences) by crossing with the FLPeR deleter mouse. The Foxb1(Cre) line showed Cre recombinase activity as well as EGFP fluorescence reproducing Foxb1 expression accurately. By crossing Foxb1(Cre) mice with the ROSA26R and Z/AP mouse reporter lines we have been able to trace the lineage of Foxb1-expressing cells. Early transient expression of Foxb1 in the paraxial mesoderm translates into labeling of the somites. In the central nervous system (CNS), the Foxb1 lineage includes the thalamus and mammillary body (hypothalamus), brainstem, and the ventral spinal cord and floor plate.
Assuntos
Sistema Nervoso Central/embriologia , Fatores de Transcrição Forkhead/genética , Integrases/genética , Mutagênese Insercional/métodos , Animais , Tronco Encefálico/embriologia , Tronco Encefálico/metabolismo , Sistema Nervoso Central/metabolismo , Diencéfalo/embriologia , Diencéfalo/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/genética , Integrases/análise , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Recombinação Genética , Somitos/embriologia , Somitos/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismoRESUMO
OBJECTIVE: To study the molecular mechanism of integron related gene transfer in biofilm and aqueous culture of P. aeruginosa by investigating the expression level of intI1 mRNA in class 1 integron positive strains. METHODS: A competitive reverse transcription-PCR (cRT-PCR) method was designed to quantify class 1 integrase mRNA production in two clinical P. aeruginosa strains called PA10 and PA39 when they were grown in biofilms and planktonic culture. In brief, competitive DNA (cDNA) was obtained via PCR from the genomic DNA of class 1 integron positive strains. Then the competitive RNA (cRNA) was amplified from the cDNA. Finally the serials diluted cRNA were mixed separately with the total RNA extracted from the biofilm and planktonic culture of P. aeruginosa and the competitive RT-PCR were conducted. After electrophoresis, the expression level of intI1 mRNA was quantified by comparing with the amount of the cDNA. RESULTS: The PA10 and PA39 strains produced intI1 mRNA both in their biofilms and planktonic cells. Furthermore the expression levels of intI1 mRNA from the two strains were of approximation in their biofilm and plankton stage respectitively, while the quantities of intI1 mRNA expression in their biofilm stage were about 15 times higher than those in their planktonic stage. CONCLUSION: The integrase gene is up-regulated at mRNA level in P. aeruginosa biofilm, which may be one of the reseans for the spread of antibiotic resistance and the formation of multidrug resistance.
Assuntos
Biofilmes , Regulação Bacteriana da Expressão Gênica , Integrases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Técnicas de Cultura , DNA Complementar/genética , Farmacorresistência Bacteriana/genética , Resistência a Múltiplos Medicamentos/genética , Integrases/análise , Integrons/genética , Pseudomonas aeruginosa/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação GenéticaRESUMO
To generate transgenic mice that express Cre-recombinase exclusively in the megakaryocytic lineage, we modified a mouse bacterial artificial chromosome (BAC) clone by homologous recombination and replaced the first exon of the platelet factor 4 (Pf4), also called CXCL4, with a codon-improved Cre cDNA. Several strains expressing the transgene were obtained and one strain, Q3, was studied in detail. Crossing Q3 mice with the ROSA26-lacZ reporter strain showed that Cre-recombinase activity was confined to megakaryocytes. These results were further verified by crossing the Q3 mice with a strain containing loxP-flanked integrin beta1. Excision of this conditional allele in megakaryocytes was complete at the DNA level, and platelets were virtually devoid of the integrin beta1 protein. The Pf4-Cre transgenic strain will be a valuable tool to study megakaryopoiesis, platelet formation, and platelet function.
Assuntos
Plaquetas/fisiologia , Linhagem da Célula , Megacariócitos/fisiologia , Fator Plaquetário 4/genética , Transgenes , Animais , Cromossomos Artificiais Bacterianos , Integrases/análise , Integrases/genética , Camundongos , Camundongos Transgênicos , Recombinação GenéticaRESUMO
Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bacteriófagos/enzimologia , Integrases/metabolismo , Proteínas Nucleares/metabolismo , Recombinação Genética , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/química , Sítios de Ligação , Linhagem Celular , Proteínas Correpressoras , Humanos , Imunoprecipitação , Inibidores de Integrase/metabolismo , Integrases/análise , Integrases/química , Chaperonas Moleculares , Proteínas Nucleares/análise , Proteínas Nucleares/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
A transgenic mouse line expressing the CreER(TM) fusion protein under the control of the Math1 enhancer was generated. Expression of the transgene in the postnatal mouse was restricted to hair cells of the inner ear and granule neurons in the external granule layer of the cerebellum in a temporally regulated manner. Cre activity was virtually nonexistent in uninduced mice; however, treatment of newborn pups with tamoxifen, leading to nuclear translocation of the fusion protein, resulted in efficient recombination at LoxP sites in the appropriate cell types. Up to two thirds of cerebellar granule neurons and 80-90% of cochlear hair cells underwent Cre-specific recombination. This mouse line provides a powerful tool to dissect gene function at early and late stages in development of the cerebellum and inner ear.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cerebelo/enzimologia , Células Ciliadas Auditivas Internas/enzimologia , Integrases/análise , Camundongos Transgênicos , Modelos Animais , Animais , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Elementos Facilitadores Genéticos , Integrases/genética , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Transgenes/genéticaRESUMO
The ability to target gene deletion to a specific cellular compartment via the Cre/loxP system has been a powerful tool in the analysis of broadly expressed genes. Here, we report the generation of a transgenic mouse line in which expression of Cre-recombinase is under the regulatory control of the VE-Cadherin promoter. Temporal distribution and activity of the enzyme was evaluated with two independent Cre reporter lines. Histological analysis was performed throughout development and in the adult. Recombination of lox P sites with subsequent expression of beta-galactosidase or GFP was detected as early as E7.5 in endothelial cells of the yolk sac. Progressive staining of the embryonic vasculature was noted from E8.5-13.5; however, more contiguous reporter expression was only seen by E14.5 onward in all endothelial compartments including arteries, veins, and capillaries. In addition, we found Cre activity in lymphatic endothelial cells. Unlike other endothelial-specific Cre mice, this model showed expression in the adult quiescent vasculature. Furthermore, the constitutive nature of the VE-Cadherin promoter in the adult can be advantageous for analysis of gene deletion in pathological settings.