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1.
PLoS One ; 16(9): e0256980, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495988

RESUMO

BACKGROUND: A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection. METHODOLOGY: This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method. RESULTS: In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection. CONCLUSIONS: This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.


Assuntos
Vacinas contra Adenovirus/imunologia , Adenovirus dos Símios/imunologia , Antígenos de Protozoários/imunologia , DNA de Protozoário/imunologia , DNA Recombinante/imunologia , Imunização Secundária/métodos , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/imunologia , Vacinas contra Adenovirus/administração & dosagem , Vacinas contra Adenovirus/efeitos adversos , Adenovirus dos Símios/genética , Adulto , Antígenos de Protozoários/genética , Linfócitos T CD8-Positivos/imunologia , DNA de Protozoário/genética , Epitopos/genética , Epitopos/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Voluntários Saudáveis , Humanos , Imunogenicidade da Vacina/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Masculino , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Adulto Jovem
2.
Sci Rep ; 11(1): 16579, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400669

RESUMO

Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs) (R-MrNV, I-MrNV, and E-MrNV) that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery.


Assuntos
Adenocarcinoma/tratamento farmacológico , Proteínas do Capsídeo/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Nodaviridae/química , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusão/genética
3.
Biosens Bioelectron ; 192: 113503, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34303138

RESUMO

The COVID-19 pandemic has unfortunately demonstrated how easily infectious diseases can spread and harm human life and society. As of writing, pandemic has now been on-going for more than one year. There is an urgent need for new nucleic acid-based methods that can be used to diagnose pathogens early, quickly, and accurately to effectively impede the spread of infections and gain control of epidemics. We developed a flap probe-based isothermal nucleic acid amplification method that is triggered by recombinant FEN1-Bst DNA polymerase, which-through enzymatic engineering-has both DNA synthesis, strand displacement and cleavage functions. This novel method offers a simpler and more specific probe-primer pair than those of other isothermal amplifications. We tested the method's ability to detect SARS-CoV-2 (both ORF1ab and N genes), rotavirus, and Chlamydia trachomatis. The limits of detection were 10 copies/µL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene, and 100 copies/µL for SARS-CoV-2 ORF1ab gene. There were no cross-reactions among 11 other common pathogens with characteristics similar to those of the test target, and the method showed 100% sensitivity and 100% specificity in clinical comparisons with RT-PCR testing. In addition to real-time detection, the endpoint could be displayed under a transilluminator, which is a convenient reporting method for point-of-care test settings. Therefore, this novel nucleic acid senor has great potential for use in clinical diagnostics, epidemic prevention, and epidemic control.


Assuntos
Técnicas Biossensoriais , Chlamydia trachomatis/isolamento & purificação , Rotavirus/isolamento & purificação , SARS-CoV-2/isolamento & purificação , COVID-19 , DNA Recombinante , DNA Polimerase Dirigida por DNA , Endonucleases Flap , Humanos , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Sensibilidade e Especificidade
4.
Radiat Res ; 196(3): 261-271, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34237141

RESUMO

To investigate the repairability of X-ray induced DNA damage, particularly non-double-strand breaks in living cells, enhanced green fluorescent protein (EGFP)-expressing plasmids X-ray irradiated and then transfected into nonirradiated human cells, MCF7 and MCF10A. Live-cell imaging of EGFP fluorescence was performed to measure the efficiency of plasmid repair in cells. The number of EGFP-expressing cells significantly decreased with increasing X-ray dose for both cell lines. The obtained kinetic curves of EGFP expression indicating plasmid repair were quantitatively compared against algebraically calculated ones based on the values of the transfected plasmids that had been treated with nicking or restriction enzymes. Then, assuming a Poisson distribution of single-strand breaks (SSBs), the number of cells carrying these nicked plasmids that could express EGFP were estimated. Our experimental results revealed considerably fewer cells expressing EGFP compared to the expected values we had calculated. These results suggest that the lower proportion of cells expressing EGFP as a measure of plasmid repair was due not only to the complex chemical structures of termini created by SSBs compared to those created by enzyme treatments, but also that base lesions or AP sites proximately arising at the strand-break termini might compromise EGFP expression. These results emphasize that radiation-induced DNA breaks are less repairable than enzymatically induced DNA breaks, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA.


Assuntos
Genes Reporter/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Plasmídeos/efeitos da radiação , Linhagem Celular , Dano ao DNA , Reparo do DNA , DNA Recombinante/efeitos da radiação , Células Epiteliais/efeitos da radiação , Genes BRCA1 , Proteínas de Fluorescência Verde/biossíntese , Humanos , Microscopia Intravital , Células MCF-7 , Microscopia de Fluorescência , Conformação de Ácido Nucleico/efeitos da radiação , Plasmídeos/genética , Imagem com Lapso de Tempo , Transfecção
5.
Taiwan J Obstet Gynecol ; 60(3): 530-533, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33966742

RESUMO

OBJECTIVE: We present a novel homozygous splice site mutation in the PIGN gene identified by whole exome sequencing and explored the genotype-phenotype correlation. CASE REPORT: A healthy 32-year-old woman underwent an ultrasound at 13 + 5 weeks of gestation. The ultrasound revealed multiple anomalies again including cystic hygroma, omphalocele and a ventricular septal defect. The pregnancy was subsequently terminated, and whole exome sequencing revealed a novel homozygous splice site mutation in the PIGN gene c.963 G > A (p.Gln321Gln). The same variant was also detected by pedigree-based Sanger sequencing in both parents as heterozygous, while they had normal karyotypes. CONCLUSION: Our case report enhances the phenotype-genotype correlation associated with homozygous loss of function mutations in the PIGN gene.


Assuntos
Anormalidades Múltiplas/diagnóstico , DNA Recombinante/genética , Mutação com Perda de Função/genética , Fosfotransferases/genética , Ultrassonografia Pré-Natal , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Aborto Eugênico , Adulto , Feminino , Estudos de Associação Genética , Homozigoto , Humanos , Linhagem , Gravidez , Sequenciamento Completo do Exoma
6.
Sheng Wu Gong Cheng Xue Bao ; 37(4): 1376-1384, 2021 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-33973450

RESUMO

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.


Assuntos
Herpesvirus Galináceo 2 , Doença de Marek , Animais , Embrião de Galinha , Galinhas , Cromossomos Artificiais Bacterianos , DNA Recombinante , Herpesvirus Galináceo 2/genética
7.
Microbiol Res ; 248: 126764, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33887535

RESUMO

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3'NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.


Assuntos
Biomimética/métodos , Cromossomos Artificiais/genética , DNA de Protozoário/genética , DNA Ribossômico/genética , Macronúcleo/genética , Tetrahymena thermophila/genética , DNA Recombinante/genética
8.
J Biosci Bioeng ; 132(1): 56-63, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33896701

RESUMO

Dissolved oxygen (DO)-stat fed-batch culture, which allows a high cell density culture of microorganisms under constant DO conditions, was applied to anti-CRP single-chain variable fragment (scFv) production using recombinant Escherichia coli. The DO-stat fed-batch culture was successfully performed under various DO conditions for more than 50 h, resulting in increased scFv production from 0.5 to 0.8 g/L by flask and batch cultures to 2.8-3.0 g/L by the fed-batch culture under the conditions of 5-40% of DO saturation. The formation of inclusion bodies was effectively depressed during DO-stat fed-batch operation; consequently, the solubility of anti-CRP scFv was significantly improved from 36-43% by the flask and batch cultures to 96-98% by the DO-stat fed-batch culture under a wide range of DO conditions. From the kinetic analysis of fed-batch experiments, it was also found that the successful folding of anti-CRP scFv in the cytoplasm occurred when metabolic rates, such as the specific growth rate and specific glucose consumption rate, were relatively low. These results show that the fed-batch culture operated by the DO-stat feeding strategy was effective for the enhanced production of anti-CRP scFv with high solubility.


Assuntos
Técnicas de Cultura Celular por Lotes , DNA Recombinante/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Anticorpos de Cadeia Única/biossíntese , Citoplasma/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Fermentação , Corpos de Inclusão/metabolismo , Cinética , Oxigênio/metabolismo
9.
Trends Genet ; 37(8): 695-698, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33892960

RESUMO

Like protein-coding genes, long noncoding RNA (lncRNA) genes are composed of introns and exons. After their transcription, lncRNAs are subject to constitutive and/or alternative splicing. Here, we describe the current knowledge on lncRNA splice variants and their functional implications in cell biology.


Assuntos
Processamento Alternativo/genética , DNA Recombinante/genética , RNA Longo não Codificante/genética , Éxons/genética , Íntrons/genética
10.
PLoS Negl Trop Dis ; 15(4): e0009377, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33905412

RESUMO

Our understanding of folate metabolism in Leishmania has greatly benefited from studies of resistance to the inhibitor methotrexate (MTX). Folates are reduced in Leishmania by the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS) and by pteridine reductase (PTR1). To further our understanding of folate metabolism in Leishmania, a Cos-seq genome-wide gain of function screen was performed against MTX and against the two thymidylate synthase (TS) inhibitors 5-fluorouracil and pemetrexed. The screen revealed DHFR-TS and PTR1 but also the nucleoside transporter NT1 and one hypothetical gene derived from chromosome 31. For MTX, the concentration of folate in the culture medium affected the enrichment pattern for genes retrieved by Cos-seq. We generated a L. infantum DHFR-TS null mutant that was thymidine auxotroph, a phenotype that could be rescued by the addition of thymidine or by transfection of the flavin dependent bacterial TS gene ThyX. In these DHFR-TS null mutants it was impossible to obtain a chromosomal null mutant of PTR1 except if DHFR-TS or PTR1 were provided episomally. The transfection of ThyX however did not allow the elimination of PTR1 in a DHFR-TS null mutant. Leishmania can survive without copies of either DHFR-TS or PTR1 but not without both. Provided that our results observed with the insect stage parasites are also replicated with intracellular parasites, it would suggest that antifolate therapy in Leishmania would only work if both DHFR-TS and PTR1 would be targeted simultaneously.


Assuntos
Deleção de Genes , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Metotrexato/farmacologia , Complexos Multienzimáticos/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Animais , DNA de Protozoário/genética , DNA Recombinante/genética , Resistência a Medicamentos , Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Leishmania infantum/enzimologia , Metotrexato/metabolismo , Complexos Multienzimáticos/metabolismo , Fenótipo , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismo , Transfecção
11.
Cells ; 10(2)2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670607

RESUMO

Several methods for the stimulation of skin wound repair have been proposed over the last few decades. The most promising among them are gene and stem cell therapy. Our present experiments combined several approaches via the application of human umbilical cord blood mononuclear cells (hUCB-MC) that were transfected with pBud-VEGF165-FGF2 plasmid (gene-cell therapy) and direct gene therapy using pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. The dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that, with two weeks post-transplantation, some transplanted cells still retained expression of the stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells were found among keratinocytes, hair follicle cells, endothelial cells, and in the derma. PCNA expression studies revealed that transplantation of transfected cells terminated proliferative processes in regenerating wounds earlier than transplantation of untransfected cells. In the direct gene therapy group, four days post-operatively, the processes of flap revascularization, while using Easy LDI Microcirculation Camera, was higher than in control wounded skin. We concluded that hUCB-MC can be used for the treatment of skin wounds and transfection these cells with VEGF and FGF2 genes enhances their regenerative abilities. We also concluded that the application of pBud-VEGF165-FGF2 plasmids is efficient for the direct gene therapy of skin wounds by stimulation of wound revascularization.


Assuntos
DNA Recombinante/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Fisiológica/genética , Plasmídeos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Feminino , Humanos , Masculino , Ratos , Ratos Wistar , Transfecção
12.
Gene ; 781: 145541, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33667607

RESUMO

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts.


Assuntos
Baculoviridae/genética , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Simulação por Computador , DNA Recombinante/genética , DNA Recombinante/metabolismo , Células HEK293 , Células HeLa , Humanos , Células Sf9 , Fatores de Transcrição/metabolismo
13.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526417

RESUMO

Many Escherichia coli expression vectors make use of the lac operon. In general, the lac operator (lacO) is located downstream from the promoter of the target gene, so that binding of the lac repressor blocks transcription initiation until lactose or the isopropyl-ß-d-thiogalactopyranoside (IPTG) analog is added. The protocol given here is intended for use with IPTG-inducible vectors. l-Arabinose-inducible systems derived from the ara operon offer an alternative to expression systems based on the lac operon; guidance for their use is also provided.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Isopropiltiogalactosídeo/farmacologia , Regiões Promotoras Genéticas , DNA Recombinante/genética , Escherichia coli/efeitos dos fármacos , Vetores Genéticos/metabolismo , Proteínas Recombinantes/metabolismo , Solubilidade
14.
J Mol Biol ; 433(9): 166896, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33639215

RESUMO

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , DNA Recombinante/genética , Edição de Genes/métodos , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Epitopos/genética , Epitopos/imunologia , Genes Virais/genética , Marcadores Genéticos/genética , Vetores Genéticos/genética , Genoma Viral/genética , Vírus Auxiliares/genética , Fusão de Membrana , Vírion/genética , Internalização do Vírus
15.
Virus Res ; 292: 198224, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33166564

RESUMO

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.


Assuntos
DNA Circular/genética , DNA Viral/genética , Engenharia Genética/métodos , Vírus da Hepatite B/genética , Hepatite B/virologia , Plasmídeos/genética , DNA Circular/química , DNA Recombinante/química , DNA Recombinante/genética , DNA Viral/química , Genoma Viral , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/química , Vírus da Hepatite B/metabolismo , Humanos , Integrases/metabolismo , Plasmídeos/metabolismo , Transfecção
16.
Cold Spring Harb Protoc ; 2020(12)2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262238

RESUMO

Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, ß-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.


Assuntos
Autorradiografia/métodos , Clonagem Molecular/métodos , DNA Recombinante/análise , Processamento de Imagem Assistida por Computador/métodos , Radioisótopos de Fósforo/análise , Filme para Raios X , DNA Recombinante/química , Humanos , Radioisótopos de Fósforo/química , Reprodutibilidade dos Testes
17.
Cold Spring Harb Protoc ; 2020(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139500

RESUMO

This protocol describes the standard, old-fashioned but reliable procedure for cloning linear DNA fragments whose ends are incompatible with each other but are compatible with those of the linearized vector.


Assuntos
Clonagem Molecular/métodos , DNA/genética , Vetores Genéticos/genética , Plasmídeos/genética , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Recombinante/análise , DNA Recombinante/genética , DNA Recombinante/isolamento & purificação , Eletroforese em Gel de Ágar/métodos , Escherichia coli/genética
18.
Cold Spring Harb Protoc ; 2020(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139501

RESUMO

This protocol describes procedures for cloning blunt-ended DNA fragments into linearized plasmid vectors. To obtain the maximum number of "correct" ligation products when cloning blunt-ended target fragments, the two components of DNA in the ligation reaction must be present at an appropriate ratio. If the molar ratio of plasmid vector to target DNA is too high, then the ligation reaction may generate an undesirable number of circular empty plasmids, both monomeric and polymeric; if too low, the ligation reaction may generate an excess of linear and circular homopolymers and heteropolymers of varying sizes, orientations, and compositions. For this reason, the orientation of the foreign DNA and the number of inserts in each recombinant clone must always be validated by restriction endonuclease mapping or some other means.


Assuntos
Clonagem Molecular/métodos , DNA/genética , Vetores Genéticos/genética , Plasmídeos/genética , Bacteriófago T4/enzimologia , Tampões (Química) , DNA Ligases/metabolismo , DNA Recombinante/genética , DNA Recombinante/isolamento & purificação , DNA Recombinante/metabolismo , Escherichia coli/genética , Proteínas Virais/metabolismo
19.
PLoS Pathog ; 16(11): e1008666, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33232376

RESUMO

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Genoma Viral/genética , Viremia , Animais , Linhagem Celular , Cromossomos Artificiais Bacterianos , Citomegalovirus/patogenicidade , DNA Recombinante , Modelos Animais de Doenças , Feminino , Fibroblastos/virologia , Humanos , Macaca mulatta , Masculino , Mutação , Fases de Leitura Aberta/genética , Filogenia , Especificidade da Espécie
20.
Eur J Pharmacol ; 887: 173596, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32979353

RESUMO

Bifidobacterium is a nonpathogenic strain of anaerobic bacteria that selectively localizes and proliferates in tumors. It has emerged as a specific carrier of anticancer proteins against malignant tumors. Claudins are tetraspanin transmembrane proteins that form tight junctions. Claudin-4 is overexpressed in certain epithelial malignant cancers. The C-terminal fragment of the Clostridium perfringens enterotoxin (C-CPE), an exotoxin without the cytotoxic domain, strongly binds to claudin-4. The C-CPE fusion toxin (C-CPE-PE23), which targets claudin-4, strongly suppresses tumor growth; however, C-CPE fusion toxins exhibit hepatic toxicity. In this study, we successfully generated a strain of Bifidobacterium longum that secreted C-CPE-PE23 (B. longum-C-CPE-PE23) and was specific to and cross reactive with human and mouse claudin-4. We evaluated the therapeutic potential of this strain against triple-negative breast cancer using a mouse model. C-CPE-PE23 decreased cell viability in a dose-dependent manner in human and mouse breast cancer cell lines. After intravenous injection, Bifidobacterium was specifically distributed in the tumors of mice bearing breast cancer tumors. Moreover, B. longum-C-CPE-PE23 significantly suppressed tumor growth in mice with breast cancer without serious side effects, such as weight loss or hepatic and renal damage. We suggest that B. longum-C-CPE-PE23 is a good candidate for breast cancer treatment. Bifidobacterium could also be used as a drug delivery system for hepatotoxic agents.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Bifidobacterium/metabolismo , Claudinas/metabolismo , Sistemas de Liberação de Medicamentos , Neoplasias de Mama Triplo Negativas/terapia , Animais , Linhagem Celular Tumoral , Claudina-4/metabolismo , DNA Recombinante , Relação Dose-Resposta a Droga , Enterotoxinas/administração & dosagem , Enterotoxinas/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética
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