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1.
Adv Exp Med Biol ; 1167: 237-248, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31520359

RESUMO

In recent years, there has been growing interest in using Drosophila for drug discovery as it provides a unique opportunity to screen small molecules against complex disease phenotypes in a whole animal setting. Furthermore, gene-compound interaction experiments that combine compound feeding with complex genetic manipulations enable exploration of compound mechanisms of response and resistance to an extent that is difficult to achieve in other experimental models. Here, I discuss how compound screening and testing approaches reported in Drosophila fit into the current cancer drug discovery pipeline. I then propose a framework for a Drosophila-based cancer drug discovery strategy which would allow the Drosophila research community to effectively leverage the power of Drosophila to identify candidate therapeutics and push our discoveries into the clinic.


Assuntos
Antineoplásicos/farmacologia , Drosophila , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Animais , Modelos Animais de Doenças , Técnicas Genéticas , Fenótipo
2.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
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
4.
Genome Biol ; 20(1): 132, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262344

RESUMO

CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However, precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an efficient and highly practical platform for DNA detection.


Assuntos
Sistemas CRISPR-Cas , DNA/análise , Técnicas Genéticas , Testes Genéticos/métodos , Escherichia coli , Humanos , Sensibilidade e Especificidade
5.
Genome Biol ; 20(1): 137, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300006

RESUMO

Systems for CRISPR-based combinatorial perturbation of two or more genes are emerging as powerful tools for uncovering genetic interactions. However, systematic identification of these relationships is complicated by sample, reagent, and biological variability. We develop a variational Bayes approach (GEMINI) that jointly analyzes all samples and reagents to identify genetic interactions in pairwise knockout screens. The improved accuracy and scalability of GEMINI enables the systematic analysis of combinatorial CRISPR knockout screens, regardless of design and dimension. GEMINI is available as an open source R package on GitHub at https://github.com/sellerslab/gemini .


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas Genéticas , Software , Teorema de Bayes , Epistasia Genética
6.
Plant Physiol Biochem ; 141: 300-305, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202194

RESUMO

MYB-type transcription factors are known to participate in the response of plants to a number of stress agents. MsMYB2L is an alfalfa member of this large gene family. Its transcription in alfalfa seedlings was found to be rapidly and strongly induced by salinity, moisture deficiency and exogenously supplied abscisic acid. An analysis based on a yeast one hybrid assay indicated that its product is able to activate transcription, consistent with its function as a transcription factor. When the gene was constitutively expressed in Arabidopsis thaliana, both germination and seedling growth were more sensitive to ABA treatment than wild type, and growth was less strongly compromised by salinity and moisture deficiency stress, presumably as a result of the induction of certain stress-related genes active in ABA-dependent pathways. The transgenic seedlings' enhanced the synthesis of many osmotic regulatory substances such as proline and soluble sugar, and decreased the lipid peroxidation. In all, MsMYB2L represents a potential candidate gene for manipulating the salinity and drought tolerance of alfalfa.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/fisiologia , Proteínas de Ligação a DNA/genética , Secas , Medicago sativa/genética , Proteínas de Plantas/genética , Salinidade , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas , Técnicas Genéticas , Germinação , Filogenia , Plantas Geneticamente Modificadas/fisiologia , Polietilenoglicóis/química , Plântula/fisiologia , Estresse Fisiológico , Açúcares/química , Transcrição Genética , Ativação Transcricional
7.
Genome Biol ; 20(1): 113, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159834

RESUMO

BACKGROUND: Genes are comprised of DNA codes and contain promoters and other control elements for reading these codes. The rapid development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has made possible the construction of a novel code-reading system with low dependency on the native control elements. RESULTS: We develop CRISPReader, a technology for controlling promoterless gene expression in a robust fashion. We demonstrate that this tool is highly efficient in controlling transcription and translation initiation of a targeted transgene. A notable feature of CRISPReader is the ability to "read" the open reading frames of a cluster of gene without traditional regulatory elements or other cofactors. In particular, we use this strategy to construct an all-in-one AAV-CRISPR-Cas9 system by removing promoter-like elements from the expression cassette to resolve the existing AAV packaging size problem. The compact AAV-CRISPR-Cas9 is also more efficient in transactivation, DNA cleavage, and gene editing than the dual-AAV vector encoding two separate Cas9 elements, shown by targeting both reporter and endogenous genes in vitro and in vivo. CONCLUSIONS: CRISPReader represents a novel approach for gene regulation that enables minimal gene constructs to be expressed and can be used in potential biomedical applications.


Assuntos
Sistemas CRISPR-Cas , Expressão Gênica , Técnicas Genéticas , Iniciação Traducional da Cadeia Peptídica , Ativação Transcricional , Animais , Dependovirus , Luciferases de Renilla , Camundongos
8.
Genome Biol ; 20(1): 131, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253178

RESUMO

BACKGROUND: Phylogenetically informed distances are commonly used in the analysis of microbiome data, and analysts have many options to choose from. Although all phylogenetic distances share the goal of incorporating the phylogenetic relationships among the bacteria, they do so in different ways and give different pictures of the relationships between the bacterial communities. RESULTS: We investigate the properties of two classes of phylogenetically informed distances: the Unifrac family, including weighted, unweighted, and generalized Unifrac, and the DPCoA family, which we introduce here. Through several lines of evidence, including a combination of mathematical, data analytic, and computational methods, we show that a major and heretofore unrecognized cleavage in the phylogenetically informed distances is the relative weights placed on the deep and shallow parts of the phylogeny. Specifically, weighted Unifrac and DPCoA place more emphasis on the deep parts of the phylogeny, while unweighted Unifrac places more emphasis on the shallow parts of the phylogeny. Both the Unifrac and the DPCoA families have tunable parameters that can be shown to control how much emphasis the distances place on the deep or shallow parts of the phylogeny. CONCLUSIONS: Our results allow for a more informed choice of distance and give practitioners more insight into the potential differences resulting from different choices of distance.


Assuntos
Técnicas Genéticas , Filogenia
9.
Parasit Vectors ; 12(1): 321, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238993

RESUMO

BACKGROUND: Ticks are important vectors of disease-causing pathogens. With the rise of resistance to chemical acaricides, alternative methods in tick control are warranted. Gene manipulation has been successful in controlling mosquitoes and mosquito-borne diseases and is now looked upon as a candidate method to control ticks and tick-borne pathogens. Our previous study has identified the actin and ferritin promoter regions in the Haemaphysalis longicornis tick. RESULTS: Here, the ferritin-derived promoter from the H. longicornis tick was characterized in silico, and the core promoter sequences and some of its important components were identified. Several truncations of the promoter region were created and inserted to a reporter plasmid to determine the important components for its activity. The activities of the truncated promoters on the Ixodes scapularis tick cell line (ISE6) were measured via a dual luciferase assay using experimental and control reporter genes. To induce the promoter's activity, transfected ISE6 cells were exposed to ferrous sulfate. The 639 nucleotides truncated promoter showed the highest activity on ISE6 cells when exposed to 1 mM ferrous sulfate. CONCLUSION: In this study, we characterized an iron-inducible tick promoter that could be a valuable tool in the development of a gene-manipulation system to control ticks and tick-borne pathogens.


Assuntos
Ferritinas/genética , Ferro/metabolismo , Ixodes/citologia , Ixodes/genética , Regiões Promotoras Genéticas , Animais , Linhagem Celular , Compostos Ferrosos/farmacologia , Técnicas Genéticas , Luciferases/genética , Transfecção
10.
Lab Anim (NY) ; 48(7): 207-216, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31217565

RESUMO

Deep phenotyping is an emerging conceptual paradigm and experimental approach aimed at measuring and linking many aspects of a phenotype to understand its underlying biology. To date, deep phenotyping has been applied mostly in cultured cells and used less in multicellular organisms. However, in the past decade, it has increasingly been recognized that deep phenotyping could lead to a better understanding of how genetics, environment and stochasticity affect the development, physiology and behavior of an organism. The nematode Caenorhabditis elegans is an invaluable model system for studying how genes affect a phenotypic trait, and new technologies have taken advantage of the worm's physical attributes to increase the throughput and informational content of experiments. Coupling of these technical advancements with computational and analytical tools has enabled a boom in deep-phenotyping studies of C. elegans. In this Review, we highlight how these new technologies and tools are digging into the biological origins of complex, multidimensional phenotypes.


Assuntos
Caenorhabditis elegans/genética , Técnicas Genéticas , Ciência dos Animais de Laboratório/métodos , Fenótipo , Animais , Técnicas Genéticas/instrumentação , Ciência dos Animais de Laboratório/instrumentação
11.
Microb Cell Fact ; 18(1): 114, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253141

RESUMO

BACKGROUND: Actinoplanes sp. SE50/110 is a natural producer of acarbose. It has been extensively studied in the last decades, which has led to the comprehensive analysis of the whole genome, transcriptome and proteome. First genetic and microbial techniques have been successfully established allowing targeted genome editing by CRISPR/Cas9 and conjugal transfer. Still, a suitable system for the overexpression of singular genes does not exist for Actinoplanes sp. SE50/110. Here, we discuss, test and analyze different strategies by the example of the acarbose biosynthesis gene acbC. RESULTS: The integrative φC31-based vector pSET152 was chosen for the development of an expression system, as for the replicative pSG5-based vector pKC1139 unwanted vector integration by homologous recombination was observed. Since simple gene duplication by pSET152 integration under control of native promoters appeared to be insufficient for overexpression, a promoter screening experiment was carried out. We analyzed promoter strengths of five native and seven heterologous promoters using transcriptional fusion with the gusA gene and glucuronidase assays as well as reverse transcription quantitative PCR (RT-qPCR). Additionally, we mapped transcription starts and identified the promoter sequence motifs by 5'-RNAseq experiments. Promoters with medium to strong expression were included into the pSET152-system, leading to an overexpression of the acbC gene. AcbC catalyzes the first step of acarbose biosynthesis and connects primary to secondary metabolism. By overexpression, the acarbose formation was not enhanced, but slightly reduced in case of strongest overexpression. We assume either disturbance of substrate channeling or a negative feed-back inhibition by one of the intermediates, which accumulates in the acbC-overexpression mutant. According to LC-MS-analysis, we conclude, that this intermediate is valienol-7P. This points to a bottleneck in later steps of acarbose biosynthesis. CONCLUSION: Development of an overexpression system for Actinoplanes sp. SE50/110 is an important step for future metabolic engineering. This system will help altering transcript amounts of singular genes, that can be used to unclench metabolic bottlenecks and to redirect metabolic resources. Furthermore, an essential tool is provided, that can be transferred to other subspecies of Actinoplanes and industrially relevant derivatives.


Assuntos
Acarbose/metabolismo , Proteínas de Bactérias/genética , Técnicas Genéticas , Vetores Genéticos/genética , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Proteínas de Bactérias/metabolismo , Edição de Genes , Vetores Genéticos/metabolismo , Genoma Bacteriano , Proteoma , Transcriptoma
12.
Genome Biol ; 20(1): 91, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31084623

RESUMO

BACKGROUND: VirtUaL ChIP-seq Analysis through Networks (VULCAN) infers regulatory interactions of transcription factors by overlaying networks generated from publicly available tumor expression data onto ChIP-seq data. We apply our method to dissect the regulation of estrogen receptor-alpha activation in breast cancer to identify potential co-regulators of the estrogen receptor's transcriptional response. RESULTS: VULCAN analysis of estrogen receptor activation in breast cancer highlights the key components of the estrogen receptor complex alongside a novel interaction with GRHL2. We demonstrate that GRHL2 is recruited to a subset of estrogen receptor binding sites and regulates transcriptional output, as evidenced by changes in estrogen receptor-associated eRNA expression and stronger estrogen receptor binding at active enhancers after GRHL2 knockdown. CONCLUSIONS: Our findings provide new insight into the role of GRHL2 in regulating eRNA transcription as part of estrogen receptor signaling. These results demonstrate VULCAN, available from Bioconductor, as a powerful predictive tool.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas Genéticas , Fatores de Transcrição/metabolismo , Algoritmos , Feminino , Humanos
13.
Genome Biol ; 20(1): 94, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097038

RESUMO

Gene co-expression networks capture biological relationships between genes and are important tools in predicting gene function and understanding disease mechanisms. We show that technical and biological artifacts in gene expression data confound commonly used network reconstruction algorithms. We demonstrate theoretically, in simulation, and empirically, that principal component correction of gene expression measurements prior to network inference can reduce false discoveries. Using data from the GTEx project in multiple tissues, we show that this approach reduces false discoveries beyond correcting only for known confounders.


Assuntos
Redes Reguladoras de Genes , Técnicas Genéticas , Artefatos , Humanos
14.
Genome Biol ; 20(1): 102, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118054

RESUMO

Capture Hi-C (CHi-C) is a new technique for assessing genome organization based on chromosome conformation capture coupled to oligonucleotide capture of regions of interest, such as gene promoters. Chromatin loop detection is challenging because existing Hi-C/4C-like tools, which make different assumptions about the technical biases presented, are often unsuitable. We describe a new approach, ChiCMaxima, which uses local maxima combined with limited filtering to detect DNA looping interactions, integrating information from biological replicates. ChiCMaxima shows more stringency and robustness compared to previously developed tools. The tool includes a GUI browser for flexible visualization of CHi-C profiles alongside epigenomic tracks.


Assuntos
Cromatina , Técnicas Genéticas , Genômica/métodos , Software
15.
Genome Biol ; 20(1): 107, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138268

RESUMO

The rapid increase of omic data has greatly facilitated the investigation of associations between omic profiles such as DNA methylation (DNAm) and complex traits in large cohorts. Here, we propose a mixed-linear-model-based method called MOMENT that tests for association between a DNAm probe and trait with all other distal probes fitted in multiple random-effect components to account for unobserved confounders. We demonstrate by simulations that MOMENT shows a lower false positive rate and more robustness than existing methods. MOMENT has been implemented in a versatile software package called OSCA together with a number of other implementations for omic-data-based analyses.


Assuntos
Técnicas Genéticas , Metabolômica/métodos , Software , Idoso , Simulação por Computador , Metilação de DNA , Humanos , Modelos Lineares , Fenótipo
16.
Genome Biol ; 20(1): 85, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036053

RESUMO

Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.


Assuntos
Enzimas de Restrição do DNA , Técnicas Genéticas , Sitios de Sequências Rotuladas , Animais , Humanos
17.
Genome Biol ; 20(1): 73, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31036069

RESUMO

Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Evolução Molecular , Técnicas Genéticas , Oryza/genética , Spliceossomos , Álcoois Graxos , Proteínas de Plantas/genética , Domínios Proteicos , Piranos
18.
New Bioeth ; 25(2): 153-171, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31130111

RESUMO

The successes of the human genome project and genomics research programs portend great potential to improve upon health and enhance life. As scientific advancements continue, bioethicists and policy makers deliberate over the social and ethical implications of genetic and genomic technologies and information (ggT/I). The application of ggT/I to human reproduction raises conceptual and moral questions about being human and the links between offspring, parents, and society. Given ggT/I's ability to significantly affect the biological constitution of humans and future human generations thinking through such issues is fundamental to ethical and policy analysis. By means of a systematic literature review and accompanying content analysis, this paper highlights the dominant ethical concerns raised within recent bioethics discourse over the use of ggT/I for human reproduction. Based on these findings it aso offers a framework through which, and demarcates where, religious perspectives can add value to genethics debates and policy deliberation.


Assuntos
Pesquisa em Genética/ética , Técnicas Genéticas/ética , Genômica/ética , Religião e Ciência , Reprodução/ética , Humanos , Obrigações Morais
19.
Mol Diagn Ther ; 23(2): 187-200, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30945167

RESUMO

Designer nucleases are versatile tools for genome modification and therapy development and have gained widespread accessibility with the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology. Prokaryotic RNA-guided nucleases of CRISPR/Cas type, since first being adopted as editing tools in eukaryotic cells, have experienced rapid uptake and development. Diverse modes of delivery by viral and non-viral vectors and ongoing discovery and engineering of new CRISPR/Cas-type tools with alternative target site requirements, cleavage patterns and DNA- or RNA-specific action continue to expand the versatility of this family of nucleases. CRISPR/Cas-based molecules may also act without double-strand breaks as DNA base editors or even without single-stranded cleavage, be it as epigenetic regulators, transcription factors or RNA base editors, with further scope for discovery and development. For many potential therapeutic applications of CRISPR/Cas-type molecules and their derivatives, efficiencies still need to be improved and safety issues addressed, including those of preexisting immunity against Cas molecules, off-target activity and recombination and sequence alterations relating to double-strand-break events. This review gives a concise overview of current CRISPR/Cas tools, applications, concerns and trends.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas Genéticas , Quebras de DNA de Cadeia Dupla , Endonucleases/metabolismo , Edição de Genes , Terapia Genética , Humanos
20.
Nat Protoc ; 14(5): 1489-1508, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962605

RESUMO

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.


Assuntos
Dano ao DNA , Reparo do DNA , RNA não Traduzido , Ribonuclease Pancreático/metabolismo , Animais , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Técnicas Genéticas , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , RNA/análise , RNA/genética , RNA/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/fisiologia
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