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2.
BMC Plant Biol ; 19(1): 371, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438856

RESUMO

BACKGROUND: Propamocarb (PM) is one of the main pesticides used for controlling cucumber downy mildew. However, due to its volatility and internal absorption, PM can easily form pesticide residues on cucumber fruits that seriously endanger human health and pollute the environment. The breeding of new cucumber varieties with a low abundance of PM residues via genetic methods constitutes an effective strategy for reducing pesticide residues and improving cucumber safety and quality. To help elucidate the molecular mechanism resulting in a low PM residue abundance in cucumber, we used the cucumber cultivar 'D0351' (which has the lowest PM residue content) as the test material and identified genes related to low PM residue abundance through high-throughput tag-sequencing (Tag-Seq). RESULTS: CsMAPEG was constitutively expressed and showed both varietal and organizational differences. This gene was strongly expressed in 'D0351'. The expression levels of CsMAPEG in different cucumber tissues under PM stress were as follows: fruit>leaf>stem>root. CsMAPEG can respond to salicylic acid (SA), gibberellin (GA) and Corynespora cassiicola Wei (Cor) stress and thus plays an important regulatory role in plant responses to abiotic and biological stresses. The PM residue abundance in the fruits of CsMAPEG-overexpressing plants was lower than those found in antisense CsMAPEG plants and wild-type plants at all tested time points. The results revealed that CsMAPEG played a positive role in reducing the PM residue abundance. A CsMAPEG sense construct increased the contents of SOD, POD and GST in cucumber fruits, enhanced the degradation and metabolism of PM in cucumber, and thus effectively reduced the pesticide residue abundance in cucumber fruits. CONCLUSIONS: The expression patterns of CsMAPEG in cucumber cultivars with high and low pesticide residue abundances and a transgenic verification analysis showed that CsMAPEG can actively respond to PM stress and effectively reduce the PM residue abundance in cucumber fruits. The results of this study will help researchers further elucidate the mechanism responsible for a low PM residue abundance in cucumber and lay a foundation for the breeding of new agricultural cucumber varieties with low pesticide residue abundances.


Assuntos
Carbamatos/farmacologia , Cucumis sativus/genética , Fungicidas Industriais/farmacologia , Genes de Plantas , Resíduos de Praguicidas , Clonagem Molecular , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformação Genética
3.
J Zoo Wildl Med ; 50(2): 478-481, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31260219

RESUMO

Red pandas (Ailurus fulgens) are susceptible to canine distemper, with a number of reported vaccine-induced canine distemper cases. Canarypox-vectored recombinant canine distemper vaccines (PureVax Ferret Distemper [PFD] and Recombitek CDV [rCDV]) provide protection without inoculating a live distemper virus, but there are currently no published data regarding these vaccines' safety and efficacy in red pandas. One hundred twenty-two serum samples were collected from 50 captive red pandas and analyzed for antibodies to canine distemper. All naïve red pandas (n = 20) had negative titers. Naïve pandas receiving two PFD vaccinations had either negative or intermediate titers (n = 4). In contrast, naïve pandas receiving a series of two or three rCDV vaccinations (n = 14) had greater antibody responses. Red pandas vaccinated with PFD >12 mo since their last vaccination and a rCDV booster vaccination showed the highest titers observed. We recommend red pandas be administered a series of at least three recombinant vaccine (PDF or rDCV) vaccinations, followed by annual booster vaccinations.


Assuntos
Ailuridae/sangue , Anticorpos Antivirais/sangue , Vírus da Cinomose Canina/imunologia , Cinomose/prevenção & controle , Vacinas Virais/imunologia , Animais , Cinomose/virologia , Vetores Genéticos , Imunização Secundária , Vacinação , Vacinas Sintéticas/imunologia
4.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1307-1316, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328487

RESUMO

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.


Assuntos
Vetores Genéticos , Lentivirus , Células HEK293 , Humanos , Transdução Genética , Transfecção
5.
BMC Plant Biol ; 19(1): 311, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307375

RESUMO

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds. RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.


Assuntos
Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Genes de Plantas , RNA Guia , Soja/genética , Ácido alfa-Linoleico/genética , Agrobacterium/genética , Sistemas CRISPR-Cas , Marcadores Genéticos , Vetores Genéticos , Técnicas de Genotipagem , Padrões de Herança , Plantas Geneticamente Modificadas
7.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(7): 708-712, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31315773

RESUMO

OBJECTIVE: To construct the recombinant adenoviral vector carrying the rat interleukin-10 (rIL-10) gene, and to investigate whether it is stably expressed in bone marrow mesenchymal stem cells. METHODS: The rIL-10 gene was amplified by PCR from template rIL-10 cDNA, and the recovered 656 bp rIL-10 DNA fragment was cloned into pcDNA3.1 to construct pcDNA3.1-IL-10. Then HEK293 cells were transfected with pcDNA3.1-IL-10 and adenoviral vector for homologous recombination, and sequencing and PCR were used to evaluate whether recombination was successful. HEK293 cells were lysed by repeated freeze-thaw cycles, and bone marrow mesenchymal stem cells were infected with the virus solution containing the rIL-10 gene. Western blot was used to measure the expression of rIL-10 in bone marrow mesenchymal stem cells. RESULTS: Sequencing and PCR verified that the rIL-10 adenoviral vector was successfully constructed, with a virus titer of 4×109 PFU/mL. The expression of IL-10 was detected after bone marrow mesenchymal stem cells were infected by the virus solution containing the rIL-10 gene. CONCLUSIONS: The constructed rIL-10 recombinant adenovirus can mediate the stable expression of rIL-10 gene in bone marrow mesenchymal stem cells, which provides a basis for gene transplantation therapy of inflammatory bowel disease.


Assuntos
Células-Tronco Mesenquimais , Adenoviridae , Animais , Células da Medula Óssea , Vetores Genéticos , Células HEK293 , Humanos , Interleucina-10 , Ratos , Transfecção
8.
J Biomed Nanotechnol ; 15(3): 431-442, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31165690

RESUMO

Human Wnt inhibitory factor-1 (hWIF-1), as an anti-oncogene, holds great promise for non-small-cell lung cancer (NSCLC) therapy. However, the clinical application of hWIF-1 in cancer therapy is limited by elimination and degradation of free hWIF-1 in vivo. Therefore, it is necessary to develop safe and effective gene delivery vectors for hWIF-1 delivery in vivo. In this paper, we synthesized a novel polyethylenimine (PEI) derivative PEI-SP5-2 (PES) based on branched PEI1800 and NSCLC-targeting peptide SP5-2 to deliver hWIF-1 for NSCLC therapy. PES had excellent gene delivery capacity, and the transfection efficiency reached 50.02% ± 4.75% in A549 cell lines when the weight ratio of PES/gene was 100. Besides, the PES/gene particles were monodispersed, and the hydrodynamic diameter and zeta potential were 47.55 nm and 24.9 mV, respectively. In addition, PES/hWIF-1 complexes could inhibit the tumor growth in vitro and in vivo when it was used for non-small-cell lung cancer therapy. We concluded that PES would be promising as a novel gene delivery vector, and PES/hWIF-1 complexes inhibited the tumor growth and showed potential for non-small-cell lung cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Polietilenoimina , Transfecção
9.
Gene ; 710: 265-272, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200085

RESUMO

Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common ß2 integrin subunit (CD18/ITGB2 gene). We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5' 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.


Assuntos
Antígenos CD18/genética , Proteínas Cromossômicas não Histona/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Lentivirus/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Fator 1 de Elongação de Peptídeos/genética , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Síndrome da Aderência Leucocítica Deficitária/terapia , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Transdução Genética
10.
Acta Virol ; 63(2): 162-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230445

RESUMO

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.


Assuntos
Fígado , Regiões Promotoras Genéticas , Spumavirus , Adulto , Animais , Linhagem Celular , Cricetinae , Terapia Genética , Vetores Genéticos , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Regiões Promotoras Genéticas/genética , Spumavirus/genética
11.
Croat Med J ; 60(3): 201-211, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31187947

RESUMO

AIM: To assess whether an adenoviral vector carrying the bone morphogenetic protein genes (Ad.BMP-2) can transduce human muscle tissue and direct it toward osteogenic differentiation within one hour. METHODS: This in vitro study, performed at the Department of Molecular Biology, Faculty of Science, Zagreb from 2012 to 2017, used human muscle tissue samples collected during anterior cruciate ligament reconstructions performed in St Catherine Hospital, Zabok. Samples from 28 patients were transduced with adenoviral vector carrying firefly luciferase cDNA (Ad.luc) by using different doses and times of transduction, and with addition of positive ions for transduction enhancement. The optimized protocol was further tested on muscle samples from three new patients, which were transduced with Ad.BMP-2. Released bone morphogenetic protein 2 (BMP-2) levels in osteogenic medium were measured every three days during a period of 21 days. Expression of osteogenic markers was measured at day 14 and 21. After 21 days of cultivation, muscle tissue was immunohistochemically stained for collagen type I detection (COL-I). RESULTS: The new transduction protocol was established using 108 plaque-forming units (P<0.001) as an optimal dose of adenoviral vector and 30 minutes (P<0.001) as an optimal contact time. Positive ions did not enhance transduction. Samples transduced with Ad.BMP-2 according to the optimized protocol showed enhanced expression of osteogenic markers (P<0.050), BMP-2 (P<0.001), and COL I. CONCLUSION: This study confirms that Ad.BMP-2 can transduce human muscle tissue and direct it toward osteogenic differentiation within 30 minutes.


Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/genética , Músculo Esquelético/fisiologia , Osteogênese/genética , Transdução Genética , Adenoviridae , Adolescente , Adulto , Células Cultivadas , Melhoramento Genético , Vetores Genéticos , Humanos , Pessoa de Meia-Idade , Tendões/fisiologia , Adulto Jovem
12.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 892-900, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223007

RESUMO

To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.


Assuntos
Proliferação de Células , Vetores Genéticos , MicroRNAs , Animais , Linhagem Celular , Proliferação de Células/genética , Células Epiteliais/citologia , MicroRNAs/genética , Suínos
13.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1135-1142, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232010

RESUMO

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²âº affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.


Assuntos
Baculoviridae , Vetores Genéticos , Animais , Humanos , Proteínas Recombinantes , Células Sf9 , Spodoptera
14.
Prep Biochem Biotechnol ; 49(8): 822-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156045

RESUMO

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.


Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Lentivirus/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Dosagem de Genes , Humanos , Proteínas Recombinantes/genética , Transdução Genética
15.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(4): 333-338, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31167693

RESUMO

Objective To obtain the expression vector, which could be used for screening peptide drug against human immunodeficiency virus type 1 (HIV-1) integrase (IN) with bimolecular fluorescence complementation (BiFc). Methods Full-length IN sequence was amplified using high-fidelity PCR with the template pMDL vector, following with the insertion of target sequence into pBiFc-VN173 vector. Moreover, the recombinant vector pBiFc-VN173-IN was further confirmed by double enzyme digestion and sequencing. Compared with empty control, expression of IN from pBiFc-VN173-IN in HEK293T cells was validated by Western blotting and immunofluorescence assay (IFA). Results The pBiFc-VN173-IN vector, which could drive the ectopic expression of IN, was successfully obtained through high-fidelity PCR, vector construction and confirmation. In addition, Western blot analysis and IFA validated the ectopic expression of IN in HEK293T cells after transfection. Conclusion The pBiFc-VN173-IN vector has been successfully obtained, and it will be helpful for screening specific peptides against IN using BiFc.


Assuntos
Vetores Genéticos , Inibidores de Integrase de HIV/farmacologia , HIV-1 , Peptídeos/farmacologia , Western Blotting , Células HEK293 , Integrase de HIV , Humanos , Transfecção
16.
Nat Commun ; 10(1): 2315, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127098

RESUMO

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Medo/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Condicionamento (Psicologia)/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Modelos Animais , Neurônios/metabolismo , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Técnicas Estereotáxicas
17.
Nat Commun ; 10(1): 2262, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118412

RESUMO

Most biomedical research aimed at understanding gene function uses the Cre-Lox system, which consists of the Cre recombinase-dependent deletion of genes containing LoxP sites. This system enables conditional genetic modifications because the expression and activity of the recombinase Cre/CreERT2 can be regulated in space by tissue-specific promoters and in time by the ligand tamoxifen. Since the precise Cre-Lox recombination event is invisible, methods were developed to report Cre activity and are widely used. However, numerous studies have shown that expression of a given Cre activity reporter cannot be assumed to indicate deletion of other LoxP-flanked genes of interest. Here, we report the generation of an inducible dual reporter-Cre mouse allele, iSuRe-Cre. By significantly increasing Cre activity in reporter-expressing cells, iSuRe-Cre provides certainty that these cells have completely recombined floxed alleles. This genetic tool increases the ease, efficiency, and reliability of conditional mutagenesis and gene function analysis.


Assuntos
Edição de Genes/métodos , Vetores Genéticos/genética , Integrases/genética , Plasmídeos/genética , Animais , Técnicas de Cultura de Células , Clonagem Molecular/métodos , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Recombinação Genética/efeitos dos fármacos , Tamoxifeno/farmacologia
18.
Methodist Debakey Cardiovasc J ; 15(1): 62-69, 2019 Jan-Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31049151

RESUMO

Cardiovascular disease is the leading cause of death worldwide, and elevated lipid levels is a major contributor. Gene delivery, which involves controlled transfer of nucleic acids into cells and tissues, has been widely used in research to study lipid metabolism and physiology. Several technologies have been developed to somatically overexpress, silence, or disrupt genes in animal models and have greatly advanced our knowledge of metabolism. This is particularly true with regard to the liver, which plays a central role in lipoprotein metabolism and is amenable to many delivery approaches. In addition to basic science applications, many of these delivery technologies have potential as gene therapies for both common and rare lipid disorders. This review discusses three major gene delivery technologies used in lipid research-including adeno-associated viral vector overexpression, antisense oligonucleotides and small interfering RNAs, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system-and examines their potential therapeutic applications.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Dislipidemias/terapia , Terapia Genética/métodos , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Animais , Sistemas CRISPR-Cas , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Dependovirus/genética , Dislipidemias/sangue , Dislipidemias/epidemiologia , Dislipidemias/genética , Edição de Genes/métodos , Terapia Genética/efeitos adversos , Vetores Genéticos , Humanos , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Resultado do Tratamento
19.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
20.
Iran J Allergy Asthma Immunol ; 18(2): 131-142, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066249

RESUMO

The Chronic granulomatous disease (CGD) is a primary immunodeficiency that characterized by mutations in phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in deficient antimicrobial activity of phagocytic cells and recurrent childhood infections. Hematopoietic stem cell transplantation (HSCT) is a curative option for patients with human leukocyte antigen (HLA) matched donor, when conventional cares and therapies fail. However, in many cases when the patients have not an HLA-matched donor, they need to a method to recapitulate the function of the affected gene within the patient's own cells. Gene therapy is a promising approach for CGD. While, the success of retroviral or lentiviral vectors in gene therapy for CGD has been hampered by random integration and insertional activation of proto-oncogenes. These serious adverse events led to improvement and generations of viral vectors with increased safety characteristics. Gene therapy continues to progress and the advent of new technologies, such as engineered endonucleases that have shown a great promise for the treatment of genetic disease. This review focuses on the application of gene therapy for the CGD, the limitations encountered in current clinical trials, advantages and disadvantages of endonucleases in gene correction and modeling with CRISPR/Cas9 approach.


Assuntos
Terapia Genética , Doença Granulomatosa Crônica/terapia , Infecção/terapia , Mutação/genética , NADPH Oxidases/genética , Animais , Sistemas CRISPR-Cas , Ensaios Clínicos como Assunto , Vetores Genéticos , Doença Granulomatosa Crônica/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Infecção/genética , Fagocitose/genética
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