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1.
Recent Results Cancer Res ; 214: 129-151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31473851

RESUMO

The adoptive cell transfer (ACT) of genetically engineered T cell receptor (TCR) T cells is one of the burgeoning fields of immunotherapy, with promising results in current clinical trials. Presently, clinicaltrials.gov has over 200 active trials involving adoptive cell therapy. The ACT of genetically engineered T cells not only allows the ability to select for TCRs with desired properties such as high-affinity receptors and tumor reactivity but to further enhance those receptors allowing for better targeting and killing of cancer cells in patients. Moreover, the addition of genetic material, including cytokines and cytokine receptors, can increase the survival and persistence of the T cell allowing for complete and sustained remission of cancer targets. The potential for improvement in adoptive cell therapy is limitless, with genetic modifications targeting to improve weaknesses of ACT and to thus enhance receptor affinity and functional avidity of the genetically engineered T cells.


Assuntos
Engenharia Genética , Imunoterapia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T , Linfócitos T/citologia , Humanos
2.
An Acad Bras Cienc ; 91(suppl 3): e20190568, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576934

RESUMO

Early humans have domesticated plant and animal species based on ancient empirical concepts (Darwin 1868, 1876). In 1886, Mendel established a new paradigm of hereditary laws (Mendel 1866, 1870, 1950) based on genotypic and phenotypic traits of cross-compatible species, establishing a complex breeding technology that is currently utilized for the development of most food and livestock-derived products. Recently, studies on deciphering the double-helical structure (Watson and Crick 1953a, b) and how to restrict DNA (Arber 2012) have established the foundation of recombinant DNA technology. A new era is paving the way for genetic manipulation of important traits among all the kingdom's organisms, allowing for the development of innovative and widely utilized products for the agricultural, industrial and pharmaceutical production sectors (Mc Elroy 2003, 2004, ISAAA 2016).


Assuntos
Biodiversidade , Conservação dos Recursos Naturais/métodos , Engenharia Genética/métodos , Criação de Animais Domésticos , Animais , Biotecnologia
3.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1662-1675, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559748

RESUMO

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.


Assuntos
Saccharopolyspora , Proteínas de Bactérias , Engenharia Genética , Inseticidas , Macrolídeos
4.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1761-1770, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559757

RESUMO

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.


Assuntos
Engenharia Genética , Escherichia coli , Proteínas de Escherichia coli , Plasmídeos , Recombinação Genética
5.
Microb Cell Fact ; 18(1): 161, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547820

RESUMO

BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.


Assuntos
Dióxido de Carbono/metabolismo , Diatomáceas/crescimento & desenvolvimento , Diatomáceas/genética , Ácidos Graxos/metabolismo , Glucosefosfato Desidrogenase/genética , Dióxido de Carbono/análise , Diatomáceas/metabolismo , Engenharia Genética , Glucosefosfato Desidrogenase/metabolismo , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fotossíntese
6.
Bioresour Technol ; 293: 122135, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31540787

RESUMO

Microalgae have attracted great attention as a promising sustainable resource for biofuel production. In studies aiming to improve lipid accumulation, many key enzymes involved in lipid biosynthesis were identified and confirmed, but genetic engineering remains a challenge in most species of microalgae. In an alternative approach, various chemical modulators can be used to directly regulate the lipid biosynthesis pathway, with similar effects to gene overexpression and interference approaches, including improving the precursor supply and blocking competing pathways. The produced lipid can be protected from being converted into other metabolites by the chemicals such as lipase inhibitors. In addition, a few chemicals were also demonstrated to greatly influence cell growth and lipid accumulation by indirect regulation of the lipid biosynthesis pathway, such as increasing cell permeability or regulating oxidative stress. Thus, adding chemical modulators can be a useful alternative strategy for improving lipid accumulation in large-scale cultivation of microalgae.


Assuntos
Microalgas , Biocombustíveis , Engenharia Genética , Lipídeos
7.
Adv Exp Med Biol ; 1148: 1-24, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482492

RESUMO

The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.


Assuntos
Enzimas/biossíntese , Enzimas/isolamento & purificação , Enzimas/farmacologia , Produtos Biológicos , Meios de Cultura , Engenharia Genética , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes
8.
Mol Plant Microbe Interact ; 32(9): 1095-1109, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31365325

RESUMO

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.


Assuntos
Cruzamento , Resistência à Doença , Engenharia Genética , Fatores de Transcrição , Verticillium , Brassica rapa/microbiologia , Resistência à Doença/genética , Mutação com Ganho de Função , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Fatores de Transcrição/genética
9.
Plant Mol Biol ; 101(3): 325-339, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31399934

RESUMO

KEY MESSAGE: Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Brassica napus/enzimologia , Engenharia Genética , Sistema de Sinalização das MAP Quinases , Fosforilação , Imunidade Vegetal , Folhas de Planta/enzimologia , Proteoma , Transdução de Sinais , Tabaco/enzimologia
10.
Bioresour Technol ; 291: 121932, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31387837

RESUMO

Microalgae and cyanobacteria are easy to culture, with higher growth rates and photosynthetic efficiencies compared to terrestrial plants, and thus generating higher productivity. The concept of microalgal biorefinery is to assimilate carbon dioxide and convert it to chemical energy/value-added products, such as vitamins, carotenoids, fatty acids, proteins and nucleic acids, to be applied in bioenergy, health foods, aquaculture feed, pharmaceutical and medical fields. Therefore, microalgae are annotated as the third generation feedstock in bioenergy and biorefinery. In past decades, many studies thrived to improve the carbon sequestration efficiency as well as enhance value-added compounds from different algae, especially via genetic engineering, synthetic biology, metabolic design and regulation. From the traditional Agrobacterium-mediated transformation DNA to novel CRISPR (clustered regularly interspaced short palindromic repeats) technology applied in microalgae and cyanobacteria, this review has highlighted the genome editing technology for biorefinery that is a highly environmental friendly trend to sustainable and renewable development.


Assuntos
Cianobactérias , Microalgas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Engenharia Genética
11.
J Agric Food Chem ; 67(35): 9749-9756, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31415718

RESUMO

Bovine lactoferrin N-lobe plays an important key in the nonimmunological defense system. In this work, the most suitable promoter Pveg was selected and the fragment coding bovine lactoferrin N-lobe was optimized according to codon bias of Bacillus. The recombinant plasmid pMA0911-Pveg-mBLF-N was introduced into Baicillus subtilis 168 to create B. subtilis/pMA0911-Pveg-mBLF-N. The bovine lactoferrin N-lobe was highly expressed at 28 °C for 15 h. Its purified protein was obtained with 16.5 mg/L and a purity of 93.6% using ammonium sulfate precipitation, Ni-NTA, and molecular exclusion. About 200 ng/mL purified bovine lactoferrin N-lobe completely inhibited cell-growth of Escherichia coli JM109 (DE3), 70.3% of Pseudomonas aeruginosa CGMCC 1.6740, and 41.5% of Staphylococcus aureus CGMCC 1.282. To our knowledge, this is the first report about active expression, purification, and characterization of bovine lactoferrin N-lobe in safe bacterium B. subtilis, which opens an available application way in the biomedical and food industries.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/genética , Códon/genética , Lactoferrina/genética , Regiões Promotoras Genéticas , Animais , Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Clonagem Molecular , Códon/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Engenharia Genética , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
14.
Microbiol Res ; 226: 1-9, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284938

RESUMO

Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.


Assuntos
Citrus/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Interferência de RNA , Bactérias/genética , Bactérias/patogenicidade , Sistemas CRISPR-Cas , Citrus/microbiologia , Produtos Agrícolas , Fungos/genética , Fungos/patogenicidade , Edição de Genes/métodos , Engenharia Genética , Vírus/genética , Vírus/patogenicidade
15.
J Agric Food Chem ; 67(30): 8393-8401, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31291721

RESUMO

The ginsenoside 20-O-ß-glucopyranosyl-20(S)-protopanaxadiol or compound K is an essential ingredient in functional food, cosmetics, and traditional medicines. However, no study has reported the complete conversion of all protopanaxadiol (PPD)-type ginsenosides from ginseng extract into compound K using whole-cell conversion. To increase the production of compound K from ginseng extract using whole recombinant cells, the ß-glucosidase enzyme from Caldicellulosiruptor bescii was coexpressed with a chaperone expression system (pGro7), and the cells expressing the coexpression system were permeabilized with ethylenediaminetetraacetic acid. The permeabilized cells carrying the chaperone coexpression system showed a 2.6-fold increase in productivity and yield as compared with nontreated cells, and completely converted all PPD-type ginsenosides from ginseng root extract into compound K with the highest productivity among the results reported so far. Our results will contribute to the industrial biological production of compound K.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Firmicutes/enzimologia , Ginsenosídeos/metabolismo , Chaperonas Moleculares/genética , Sapogeninas/metabolismo , beta-Glucosidase/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Escherichia coli/química , Firmicutes/genética , Engenharia Genética , Ginsenosídeos/química , Chaperonas Moleculares/metabolismo , Panax/química , Sapogeninas/química , beta-Glucosidase/metabolismo
16.
J Agric Food Chem ; 67(29): 8186-8190, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31272146

RESUMO

Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Indolacéticos/metabolismo , Vias Biossintéticas , Engenharia Genética , Glucose/metabolismo , Triptofano/metabolismo
17.
J Agric Food Chem ; 67(28): 7986-7994, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31282158

RESUMO

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of ß-carotene and other provitamin A carotenoids in the grain. Except for ß-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89-113% and 57-99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.


Assuntos
Oryza/genética , Plantas Geneticamente Modificadas/química , Sementes/química , Aminoácidos/análise , Aminoácidos/metabolismo , Bangladesh , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Alimentos Geneticamente Modificados , Engenharia Genética , Valor Nutritivo , Oryza/química , Oryza/metabolismo , Filipinas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Provitaminas/análise , Provitaminas/metabolismo , Sementes/genética , Sementes/metabolismo , Vitamina A/análise , Vitamina A/metabolismo , beta Caroteno/análise , beta Caroteno/metabolismo
18.
Nat Commun ; 10(1): 2960, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273196

RESUMO

Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Células Clonais , Biblioteca Gênica , Engenharia Genética , Genoma Fúngico , Proteínas de Fluorescência Verde/metabolismo , Proteínas Nucleares/metabolismo
19.
Hematol Oncol ; 37 Suppl 1: 95-100, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31187533

RESUMO

Chimeric antigen receptor (CAR) T-cell therapy has dramatically shifted the landscape of treatment for lymphoid malignancies, especially diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). However, there continue to be significant limitations of this therapy, such as incomplete or nonsustained responses and severe toxicities in a subset of patients. Furthermore, expanding the role of CAR T-cell therapy to new disease types is an important next step. In this review, we will highlight landmark trials for anti-CD19 CAR T cells and first-in-human trials of novel CARs, as well as discuss promising innovative CAR designs that are still undergoing preclinical development. Lastly, we will discuss toxicity and mechanisms of CAR T-cell resistance and failure, as well as potential future treatment approaches to these common issues.


Assuntos
Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD19 , Antígenos de Neoplasias/imunologia , Ensaios Clínicos como Assunto , Engenharia Genética , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Neoplasias/diagnóstico , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Pesquisa , Resultado do Tratamento
20.
BMC Plant Biol ; 19(1): 246, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182023

RESUMO

BACKGROUND: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. RESULTS: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. CONCLUSION: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.


Assuntos
Agrobacterium/fisiologia , Brassica rapa/genética , Brassica rapa/microbiologia , Engenharia Genética/métodos , Transformação Genética , Southern Blotting , Flores/genética , Reação em Cadeia da Polimerase
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