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1.
Appl Microbiol Biotechnol ; 104(5): 2125-2135, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932895

RESUMO

Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3' ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. • A more consistent effect was observed for methylated siRNAs. • The findings are important for development of plant gene regulation approaches.


Assuntos
Regulação da Expressão Gênica de Plantas , Inativação Gênica , RNA Interferente Pequeno/genética , Transgenes/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Metilação de DNA , Regulação da Expressão Gênica de Plantas/genética , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Genética
2.
Nat Protoc ; 14(12): 3538-3553, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31748752

RESUMO

The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.


Assuntos
Engenharia Genética/métodos , Terapia Genética/métodos , MicroRNAs/síntese química , Animais , Clonagem Molecular/métodos , Vetores Genéticos/genética , Humanos , MicroRNAs/genética , Transgenes/genética
3.
World J Gastroenterol ; 25(39): 5961-5972, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31660033

RESUMO

BACKGROUND: Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM: To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS: We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS: The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION: Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.


Assuntos
Antivirais/farmacologia , Vetores Genéticos/genética , Vírus da Hepatite B/genética , Luciferases/genética , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Genes Reporter/genética , Células Hep G2 , Humanos , Lamivudina/farmacologia , Testes de Sensibilidade Microbiana/métodos , Plasmídeos/genética , RNA/genética , RNA Viral/genética , Transfecção/métodos , Transgenes/genética , Replicação Viral/genética
4.
Nat Med ; 25(10): 1505-1511, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31591596

RESUMO

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.


Assuntos
Terapia Genética , Distrofias Musculares/terapia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Utrofina/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Cães , Distrofina/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Contração Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Transgenes/genética , Utrofina/uso terapêutico
5.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502562

RESUMO

The success of viral vectors mediated gene therapy is still hampered by immunogenicity and insufficient transgene expression. Alternatively, non-viral vectors mediated gene delivery has the advantage of low immunogenicity despite showing low transgene expression. By carefully considering the advantages of each approach, hybrid vectors are currently being developed by modifying the viral vectors using non-viral biopolymers. This review provides an overview of the hybrid vectors currently being developed.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/uso terapêutico , Animais , Biopolímeros/química , Biopolímeros/genética , Dependovirus/química , Vetores Genéticos/química , Humanos , Transgenes/genética
6.
PLoS Biol ; 17(8): e3000374, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393866

RESUMO

A deep understanding of how regulation of the multiple levels of gene expression in mammalian tissues give rise to complex phenotypes has been impeded by cellular diversity. A handful of techniques were developed to tag-select nucleic acids of interest in specific cell types, thereby enabling their capture. We expanded this strategy by developing the Tagger knock-in mouse line bearing a quad-cistronic transgene combining enrichment tools for nuclei, nascent RNA, translating mRNA, and mature microRNA (miRNA). We demonstrate that Tagger can capture the desired nucleic acids, enabling multiple omics approaches to be applied to specific cell types in vivo using a single transgenic mouse line.


Assuntos
Perfilação da Expressão Gênica/métodos , Ácidos Nucleicos/isolamento & purificação , Sequenciamento Completo do Genoma/métodos , Animais , Clonagem Molecular/métodos , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Técnicas de Introdução de Genes , Genômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , MicroRNAs/genética , Proteômica/métodos , RNA Mensageiro/genética , Transcriptoma/genética , Transgenes/genética
7.
Nucleic Acids Res ; 47(15): 8126-8135, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31429873

RESUMO

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target-DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.


Assuntos
Proteína 9 Associada à CRISPR/genética , Elementos de DNA Transponíveis/genética , Proteínas Recombinantes de Fusão/genética , Transposases/genética , Sequência de Bases , Sítios de Ligação/genética , Proteína 9 Associada à CRISPR/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Mutagênese Insercional , Proteínas Recombinantes de Fusão/metabolismo , Transgenes/genética , Transposases/metabolismo
8.
BMC Genomics ; 20(1): 594, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324144

RESUMO

BACKGROUND: The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant's genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopharmaceutical proteins. RESULTS: Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein -encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source. CONCLUSIONS: This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world.


Assuntos
Perfilação da Expressão Gênica , Genômica , Glicoproteínas/genética , Engenharia de Proteínas , Tabaco/genética , Variação Genética , Anotação de Sequência Molecular , Transgenes/genética
9.
Nature ; 571(7763): 107-111, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31217582

RESUMO

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.


Assuntos
Diarreia/congênito , Diarreia/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Intestinos/fisiologia , Deleção de Sequência/genética , Animais , Cromossomos Humanos Par 16/genética , Modelos Animais de Doenças , Feminino , Genes Reporter , Loci Gênicos/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Linhagem , Fenótipo , Ativação Transcricional , Transcriptoma/genética , Transgenes/genética
10.
BMC Biotechnol ; 19(1): 35, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208395

RESUMO

BACKGROUND: Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing ß cells. However, few studies have used vectors derived from « simple ¼ retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into ß cells. RESULTS: We here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian ß cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in ß over non-ß cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human ß cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human ß cell lines. CONCLUSION: Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian ß cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of ß cell function and monitor changes in their differentiation status.


Assuntos
Vetores Genéticos/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Luminescentes/metabolismo , Retroviridae/metabolismo , Transdução Genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Expressão Gênica , Vetores Genéticos/genética , Células HEK293 , Humanos , Células Secretoras de Insulina/citologia , Proteínas Luminescentes/genética , Camundongos , Ratos , Retroviridae/classificação , Retroviridae/genética , Transgenes/genética
11.
Genes (Basel) ; 10(6)2019 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181711

RESUMO

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.


Assuntos
Adenoviridae/isolamento & purificação , Doping nos Esportes , Vetores Genéticos/isolamento & purificação , Transgenes/genética , Adenoviridae/genética , Animais , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Camundongos
12.
Nat Plants ; 5(5): 505-511, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036912

RESUMO

The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.


Assuntos
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Troca/genética , Engenharia de Proteínas , Transgenes/genética , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética
13.
Nat Plants ; 5(5): 486-490, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036913

RESUMO

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.


Assuntos
Proteínas de Plantas/genética , Plastídeos/genética , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética , Transgenes/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Fluorescência Verde/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética , Zea mays/genética , Zea mays/metabolismo
14.
Nat Plants ; 5(5): 539-550, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31076735

RESUMO

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.


Assuntos
Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Membrana/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Interferência de RNA , RNA Nucleotidiltransferases/fisiologia , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transgenes/genética
15.
Mol Biol Rep ; 46(4): 4645-4660, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31098805

RESUMO

The discovery of small RNAs has offered exciting opportunities in manipulating gene expression. The non-coding RNAs cause target gene inactivation at the transcriptional, post-transcriptional or translational level. In addition to the default silencing approach, they provide another mode of gene regulation by transitivity. Here, gradual amplification in effector RNAs number allows regulation of genes other than the original target and causes the outspread of silencing from its origin to aid a robust response. Unlike the short-range cell-to-cell movement of silencing signal (through plasmodesmata), little is known of the mediators of systemic silencing (usually through phloem). Through the present review, we combine the reports available so far to better understand the characteristics of secondary silencing, factors involved, and summarize the instances where it has been employed in plants. Understanding the molecular mechanism behind transitivity has led to the designing of efficient transgenes for targeted gene inactivation, utilized in silencing of a multigene family, and in the field of functional genomics. Studies uncovering the origin of distinct secondary silencing pathways in plants have been exploited for developing artificial RNA silencing methods such as hairpin RNA, artificial microRNA, intrinsic direct repeat, inverted repeat, artificial trans-acting siRNA, phased siRNA, and virus-induced gene silencing. The techniques have facilitated the spread of traits such as pathogenic resistance or alter plant growth and development features. The mechanism of reprogramming in the silencing machinery and the consequent genetic manipulation through transitive RNA is still not completely understood and its exploitation in crop improvement programmes is still in a developing phase.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Interferência de RNA/fisiologia , RNA de Plantas/genética , Inativação Gênica/fisiologia , Técnicas Genéticas , MicroRNAs/genética , Plantas Geneticamente Modificadas/genética , RNA Interferente Pequeno/genética , Transgenes/genética
16.
Sensors (Basel) ; 19(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096615

RESUMO

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.


Assuntos
Técnicas Biossensoriais , Citocromo P-450 CYP1A1/isolamento & purificação , Citocromo P-450 CYP2B6/isolamento & purificação , Citocromo P-450 CYP3A/isolamento & purificação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Proteínas de Fluorescência Verde/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Taxa de Depuração Metabólica , Transgenes/genética
17.
Eur J Pharmacol ; 855: 56-64, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31034821

RESUMO

Cyclic AMP (cAMP) is an important second messenger that mediates various biological functions in both prokaryotes and eukaryotes. Due to the ever increasing significance in studying the function and modulation of cAMP-based signaling, it is important to develop a protein-based biosensor that reports the cAMP mediated gene expression. Based on a synthetic transgene approach, an artificial mammalian transactivator was developed by fusing a transcriptional regulatory element cAMP receptor protein (CRP) of Escherichia coli to the VP16 transactivation domain of Herpes simplex virus in a mammalian expression vector (pLA1) that activates CRP specific operator site present in a chimeric promoter (OCRP- PhCMVmin- Luciferase) in a concentration dependent manner in mammalian cells. Our results reveal that the engineered transactivator report the gene expression mediated by cAMP directly in mammalian cells and this cAMP reporter system works irrespective of Protein kinase A (PKA) - cyclic AMP response element binding protein (CREB) - cyclic AMP response element (CRE) signaling since the luciferase activity mediated by synthetic gene construct is seen even in the presence of PKA inhibitor H-89 (derived from H-8 (N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide). Furthermore this synthetic transcription factor plays a significant role in reporting and mediating cAMP signaling in tumorigenic cells which possess an aberrant cAMP signaling due to PKA and CREB mutations.


Assuntos
AMP Cíclico/metabolismo , Genes Reporter/genética , Engenharia Genética/métodos , Transgenes/genética , Animais , Linhagem Celular , Proteína Receptora de AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Transdução de Sinais , Transcrição Genética
18.
Genetics ; 212(2): 489-508, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30948430

RESUMO

Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented in Drosophila where homologs are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus in Drosophila and have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologs. Homotypic trans-interactions between four different insulators can support transvection: the gypsy insulator (GI), Wari, Fab-8 and 1A2; GI and Fab-8 are more effective than Wari or 1A2 We show that, in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homolog pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair in cis, and it is further suppressed by cis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The identity of enhancers and promoters in the vicinity of a trans-interacting insulator pair is also important, indicative of complex insulator-enhancer-promoter interactions.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Elementos Isolantes/genética , Regiões Promotoras Genéticas , Transgenes/genética , Animais , Cromatina/genética , Drosophila/genética , Genes Reporter/genética , Heterozigoto
19.
J Biosci Bioeng ; 128(3): 355-364, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30962099

RESUMO

To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine.


Assuntos
Regulação da Expressão Gênica , Resposta ao Choque Térmico/genética , Temperatura Alta , Nanopartículas de Magnetita , Ativação Transcricional/efeitos dos fármacos , Transgenes , Animais , Ferritinas/genética , Ferritinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Resposta ao Choque Térmico/efeitos dos fármacos , Calefação , Células Hep G2 , Humanos , Magnetismo , Biologia Sintética/métodos , Engenharia Tecidual/métodos , Transfecção/métodos , Transgenes/efeitos dos fármacos , Transgenes/genética
20.
BMC Biotechnol ; 19(1): 23, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31014302

RESUMO

BACKGROUND: The gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors. RESULTS: Four fiber-modified human adenovirus 5 (HAdV-5) vectors were investigated to transduce hematopoietic cells. F35-EG or F11p-EG were HAdV-35 or HAdV-11p fiber pseudotyped HAdV-5, and HR-EG or CR-EG vectors were generated by incorporating RGD motif to the HI loop or to the C-terminus of F11p-EG fiber. All vectors could transduce more than 90% of K562 or Jurkat cells at an multiplicity of infection (MOI) of 500 viral particle per cell (vp/cell). All vectors except HR-EG could transduce nearly 90% cord blood CD34+ cells or 80% primary human T cells at the MOI of 1000, and F11p-EG showed slight superiority to F35-EG and CR-EG. Adenoviral vectors transduced CD4+ T cells a little more efficiently than they did to CD8+ T cells. These vectors showed no cytotoxicity at an MOI as high as 1000 vp/cell because the infected and uninfected T cells retained the same CD4/CD8 ratio and cell growth rate. CONCLUSIONS: HAdV-11p fiber pseudotyped HAdV-5 could effectively transduce human T cells when human EF1a promoter was used to control the expression of transgene, suggesting its possible application in T cell immunocellular therapy.


Assuntos
Adenovírus Humanos/genética , Técnicas de Transferência de Genes/normas , Vetores Genéticos/genética , Linfócitos T/metabolismo , Proteínas da Cauda Viral/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Proliferação de Células/genética , Terapia Genética/métodos , Células HL-60 , Humanos , Células Jurkat , Células K562 , Linfócitos T/virologia , Transdução Genética/normas , Transgenes/genética , Células U937 , Proteínas da Cauda Viral/metabolismo
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