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1.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204265

RESUMO

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.


Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética
2.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 2116-2126, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227298

RESUMO

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 µg/mL RFP increased significantly, with the highest up to 842.9 µg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.


Assuntos
Espiramicina , Streptomyces , Sistemas CRISPR-Cas/genética , Engenharia Genética , Ribossomos , Streptomyces/genética
3.
Nat Commun ; 12(1): 3388, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099676

RESUMO

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.


Assuntos
Proteínas de Bactérias/efeitos da radiação , Diabetes Mellitus Tipo 2/terapia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Optogenética/métodos , Transativadores/efeitos da radiação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Celular , Diabetes Mellitus Tipo 2/genética , Feminino , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Humanos , Luz , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Obesos , Optogenética/instrumentação , Fotopletismografia/instrumentação , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Thermus thermophilus/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes , Dispositivos Eletrônicos Vestíveis
4.
Nat Commun ; 12(1): 3378, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099713

RESUMO

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.


Assuntos
Resistência à Doença/genética , Proteínas NLR/genética , Plantas Geneticamente Modificadas/microbiologia , Puccinia/patogenicidade , Triticum/microbiologia , Cromossomos de Plantas/genética , Genes de Plantas , Engenharia Genética , Marcadores Genéticos , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Caules de Planta/microbiologia , Plantas Geneticamente Modificadas/genética , Puccinia/isolamento & purificação , Triticum/genética
5.
Nat Commun ; 12(1): 3380, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099714

RESUMO

Plant-parasitic nematodes (PPNs) are economically important pests of agricultural crops, and soybean cyst nematode (SCN) in particular is responsible for a large amount of damage to soybean. The need for new solutions for controlling SCN is becoming increasingly urgent, due to the slow decline in effectiveness of the widely used native soybean resistance derived from genetic line PI 88788. Thus, developing transgenic traits for controlling SCN is of great interest. Here, we report a Bacillus thuringiensis delta-endotoxin, Cry14Ab, that controls SCN in transgenic soybean. Experiments in C. elegans suggest the mechanism by which the protein controls nematodes involves damaging the intestine, similar to the mechanism of Cry proteins used to control insects. Plants expressing Cry14Ab show a significant reduction in cyst numbers compared to control plants 30 days after infestation. Field trials also show a reduction in SCN egg counts compared with control plants, demonstrating that this protein has excellent potential to control PPNs in soybean.


Assuntos
Toxinas de Bacillus thuringiensis/genética , Produtos Agrícolas/parasitologia , Resistência à Doença/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Soja/parasitologia , Tylenchoidea/patogenicidade , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Bioensaio , Caenorhabditis elegans , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Endotoxinas/metabolismo , Feminino , Engenharia Genética , Proteínas Hemolisinas/metabolismo , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Soja/genética , Soja/metabolismo , Tylenchoidea/isolamento & purificação
6.
Nat Commun ; 12(1): 3568, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117255

RESUMO

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models.


Assuntos
Transplante de Medula Óssea , Medula Óssea/metabolismo , Quimera/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Células da Medula Óssea , Técnicas de Transferência de Genes , Engenharia Genética , Terapia Genética , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quimeras de Transplante
7.
Int J Mol Sci ; 22(9)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065074

RESUMO

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Biomarcadores , Bovinos , Técnicas de Cultura de Células , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Reprogramação Celular , Técnicas de Reprogramação Celular , Engenharia Genética , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo
9.
Stem Cell Res Ther ; 12(1): 350, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34134774

RESUMO

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Given their ability to rapidly and effectively recognize and kill aberrant cells, especially transformed cells, NK cells represent a unique cell type to genetically engineer to improve its potential as a cell-based therapy. NK cells do not express a T cell receptor and thus do not contribute to graft-versus-host disease, nor do they induce T cell-driven cytokine storms, making them highly suited as an off-the-shelf cellular therapy. The clinical efficacy of NK cell-based therapies has been hindered by limited in vivo persistence and the immunosuppressive tumor microenvironment characteristic of many cancers. Enhancing NK cell resistance to tumor inhibitory signaling through genome engineering has the potential to improve NK cell persistence in the tumor microenvironment and restore cytotoxic functions. Alongside silencing NK cell inhibitory receptors, NK cell killing can be redirected by the integration of chimeric antigen receptors (CARs). However, NK cells are associated with technical and biological challenges not observed in T cells, typically resulting in low genome editing efficiencies. Viral vectors have achieved the greatest gene transfer efficiencies but carry concerns of random, insertional mutagenesis given the high viral titers necessary. As such, this review focuses on nonviral methods of gene transfer within the context of improving cancer immunotherapy using engineered NK cells.


Assuntos
Engenharia Genética , Neoplasias , Humanos , Imunoterapia , Imunoterapia Adotiva , Células Matadoras Naturais , Neoplasias/genética , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Microambiente Tumoral
10.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072732

RESUMO

CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having unprecedented efficacy against hematological malignancies, the treatment is far from flawless. Its greatest drawbacks arise from a challenging and expensive production process, strict patient eligibility criteria and serious toxicity profile. One possible solution, supported by robust research, is the replacement of T lymphocytes with NK cells for CAR expression. NK cells seem to be an attractive vehicle for CAR expression as they can be derived from multiple sources and safely infused regardless of donor-patient matching, which greatly reduces the cost of the treatment. CAR-NK cells are known to be effective against hematological malignancies, and a growing number of preclinical findings indicate that they have activity against non-hematological neoplasms. Here, we present a thorough overview of the current state of knowledge regarding the use of CAR-NK cells in treating various solid tumors.


Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Técnicas de Cultura de Células , Ensaios Clínicos como Assunto , Terapia Combinada/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Engenharia Genética , Humanos , Células Matadoras Naturais/metabolismo , Neoplasias/diagnóstico , Neoplasias/etiologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Resultado do Tratamento
11.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071849

RESUMO

Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.


Assuntos
Engenharia Genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas , Sequência de Bases , Regulação Fúngica da Expressão Gênica , Genes Reporter , Mutação , TATA Box , Sítio de Iniciação de Transcrição
12.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066387

RESUMO

Salinity is one of the major abiotic stresses that inhibit the growth, development, and productivity of crops, particularly in hot and dry areas of the world [...].


Assuntos
Adaptação Fisiológica/genética , Plantas/genética , Estresse Salino/genética , Agricultura , Produtos Agrícolas/genética , Produtos Agrícolas/fisiologia , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Melhoramento Vegetal
13.
Molecules ; 26(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067773

RESUMO

BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.


Assuntos
Mucosa Bucal/metabolismo , Antissépticos Bucais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Reatores Biológicos/microbiologia , DNA/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Géis/farmacologia , Engenharia Genética/métodos , Humanos , Mucosa Bucal/efeitos dos fármacos , Antissépticos Bucais/metabolismo , RNA/farmacologia
14.
Molecules ; 26(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069009

RESUMO

Flavonoids are important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L-1, an induction temperature of 28 °C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caffeic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Flavonoides/biossíntese , Oxigenases de Função Mista/metabolismo , Biocatálise , Cromatografia Líquida de Alta Pressão , Escherichia coli/crescimento & desenvolvimento , Engenharia Genética , Hidroxilação , Especificidade por Substrato , Temperatura , Fatores de Tempo
15.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064885

RESUMO

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Deleção de Genes , Integrases/metabolismo , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Transgenes , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Vetores Genéticos , Integrases/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Recombinação Genética , Reprodução
16.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064900

RESUMO

Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available (Sleeping Beauty, piggyBac, and Tol2), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.


Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Engenharia Genética , Vetores Genéticos , Genoma , Transposases/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Humanos , Transposases/genética
18.
Appl Microbiol Biotechnol ; 105(12): 5067-5075, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34131780

RESUMO

Phage recombinase function unit (PRFU) plays a key role in the life cycle of phage. Repurposing this system such as lambda-Redαß or Rac-RecET for recombineering has gained success in Escherichia coli. Previous studies have showed that most PRFUs only worked well in its native hosts but poorly in the distant species. Thus, identification of new PRFUs in specific species is necessary for the development of its corresponding genetic engineering tools. Here, we present a thorough study of PRFUs in the genomes of genus Corynebacterium. We first used a database to database searching method to facilitate accurate prediction of novel PRFUs in 423 genomes. A total number of 60 sets of unique PRFUs were identified and divided into 8 types based on evolution affinities. Recombineering ability of the 8 representative PRFUs was experimentally verified in the Corynebacterium glutamicum ATCC 13032 strain. In particular, PRFU from C. aurimucosum achieved highest efficiency in both ssDNA and dsDNA mediated recombineering, which is expected to greatly facilitate genome engineering in genus Corynebacterium. These results will provide new insights for the study and application of PRFUs. KEY POINTS: • First report of bioinformatic mining and systematic analysis of Phage recombinase function unit (PRFU) in Corynebacterium genomes. • Recombineering ability of the representative PRFUs was experimentally verified in Corynebacterium glutamicum ATCC 13032 strain. • PRFU with the highest recombineering efficiency at 10-2 magnitude was identified from Corynebacterium aurimucosum.


Assuntos
Bacteriófagos , Corynebacterium glutamicum , Corynebacterium , Corynebacterium glutamicum/genética , Engenharia Genética , Recombinases
19.
GM Crops Food ; 12(1): 376-381, 2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34107854

RESUMO

Despite over 25 years of safe deployment of genetically engineered crops, the number, complexity, and scope of regulatory studies required for global approvals continue to increase devoid of adequate scientific justification. Recently, there have been calls to further expand the scope of study and data requirements to improve public acceptance. However, increased regulation can actually generate consumer distrust due to the misperception that risks are high. We believe risk-disproportionate regulation as a means to advocate for acceptance of technology is counterproductive, even though some regulatory authorities believe it part of their mandate. To help avoid public distrust, the concept of regulatory transparency to demystify regulatory decision-making should be extended to clearly justifying specific regulatory requirements as: 1) risk-driven (i.e., proportionately addressing increased risk compared with traditional breeding), or 2) advocacy-driven (i.e., primarily addressing consumer concerns and acceptance). Such transparency in the motivation for requiring risk-disproportionate studies would: 1) lessen over-prescriptive regulation, 2) save public and private resources, 3) make beneficial products and technologies available to society sooner, 4) reduce needless animal sacrifice, 5) improve regulatory decision-making regarding safety, and 6) lessen public distrust that is generated by risk-disproportionate regulation.


Assuntos
Produtos Agrícolas , Melhoramento Vegetal , Animais , Produtos Agrícolas/genética , Engenharia Genética , Plantas Geneticamente Modificadas
20.
Plant Physiol Biochem ; 164: 260-278, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34020167

RESUMO

Heavy metal (HM) accumulation in the agricultural soil and its toxicity is a major threat for plant growth and development. HMs disrupt functional integrity of the plants, induces altered phenological and physiological responses and slashes down qualitative crop yield. Chemical messengers such as phytohormones, plant growth regulators and gasotransmitters play a crucial role in regulating plant growth and development under metal toxicity in plants. Understanding the intricate network of these chemical messengers as well as interactions of genes/metabolites/proteins associated with HM toxicity in plants is necessary for deciphering insights into the regulatory circuit involved in HM tolerance. The present review describes (a) the role of chemical messengers in HM-induced toxicity mitigation, (b) possible crosstalk between phytohormones and other signaling cascades involved in plants HM tolerance and (c) the recent advancements in biotechnological interventions including genetic engineering, genome editing and omics approaches to provide a step ahead in making of improved plant against HM toxicities.


Assuntos
Metais Pesados , Engenharia Genética , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas , Plantas/genética
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