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Molecular Biology Tools to Boost the Production of Natural Products
Published in Luzia Valentina Modolo, Mary Ann Foglio, Brazilian Medicinal Plants, 2019
Luzia Valentina Modolo, Samuel Chaves-Silva, Thamara Ferreira da Silva, Cristiane Jovelina da-Silva
Genome editing is a resource used to make changes in specific regions of a genome (e.g. insertion, substitution or deletion of DNA fragments) of a cell or organism for several purposes. This technique is based on the cleavage of the DNA double strand in targeted regions followed by the use of the own cell repair system to introduce precise mutations (Tan et al., 2018). In this scenario, reverse genetics (from gene to mutant phenotype) arises as a powerful tool to unravel gene function (Alonso and Ecker, 2006). In reverse genetics, a specific gene or gene product is disrupted or modified, and the plant phenotype is consequently determined (Figure 4.1; Tierney and Lamour, 2005). Some strategies of reverse genetics are described in the literature. Among them are included those that rely on the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homologous recombination, RNAi, T-DNA insertional mutagenesis, targeting induced local lesions in genome (TILLING) and clustered, regularly interspaced, short palindromic repeat-Cas9 (CRISPR-Cas9) (Abbai et al., 2017).
Approaches to Studying Polycystic Kidney Disease in Zebrafish
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Reverse genetics, including knockdown and knockout, are widely used in studying the function of genes. In the zebrafish field, as the RNAi technique is not working, we use morpholinos (MO), which are DNA analogues and knockdown gene function by blocking translation or splicing of the target transcripts. Although this technique has been recently replaced by the CRISPR/Cas9-mediated gene knockout technique, due to the off-target issue by the MO, it is still widely used. For example, if we want to block the maternal deposit of a gene transcript and the maternal-zygotic mutant is difficult to obtain, we use MO. To avoid the nonspecific effects of the MO, we should follow the guidelines.20 Basically, the criteria for a reliable MO are that the phenotypes caused by MO knockdown should phenocopy the mutants and can be rescued by its mRNA overexpression.
Rotavirus
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Lijuan Yuan, Tammy Bui, Ashwin Ramesh
Until recently, several reverse genetics systems have been developed with only limited success, due to their requirement of a helper virus and strong selection conditions.62 An entirely plasmid-based reverse genetics approach has now been developed.257 The new strategy required coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. This new advancement in RV reverse genetics systems will facilitate improving the understanding of RV biology and pathogenesis and foster the development of RV vaccines and therapeutics. The development of RV-norovirus dual vaccines using the entirely plasmid-based reverse genetics is currently being explored.
Generation and characterization of fruitless P1 promoter mutant in Drosophila melanogaster
Published in Journal of Neurogenetics, 2021
Megan C. Neville, Alexander Eastwood, Aaron M. Allen, Ammerins de Haan, Tetsuya Nojima, Stephen F. Goodwin
While forward genetic screens remain one of the most powerful tools to study biological pathways in Drosophila, the development of reverse genetic approaches, like homologous recombination, permitted the generation or rescue of mutations in genes for which a DNA clone or sequence was available (Rong & Golic, 2000). In 2005, the Dickson lab used homologous recombination to introduce mutations in the fru locus which altered the ability of fru P1 transcripts to be sex-specifically spliced, forcing FruM expression in females; these females had been masculinized by FruM and were able to display many male pre-copulatory courtship behaviors (Demir & Dickson, 2005). Such reverse genetic approaches became more accessible with the advent of CRISPR/Cas-9 technologies, enabling genomic engineering of precise mutations in D. melanogaster with relative ease (Bassett, Tibbit, Ponting, & Liu, 2013; Gratz et al., 2013; Yu et al., 2013).
Pharmacogenomics in the era of next generation sequencing – from byte to bedside
Published in Drug Metabolism Reviews, 2021
Laura E. Russell, Yitian Zhou, Ahmed A. Almousa, Jasleen K. Sodhi, Chukwunonso K. Nwabufo, Volker M. Lauschke
Early successes of pharmacogenomics were made possible using forward genetics, in which studies aimed to identify genetic differences that might explain a given phenotype. However, this approach proves difficult for rare phenotypes and for complex genetic associations that comprise a multitude of variants with individually small effect sizes. Recent advances in sequencing technologies have opened new possibilities for reverse genetics, in which large-scale genetic data forms the basis for functional studies. In this review, we provide an updated overview of current pharmacogenetic biomarkers of clinical relevance, highlight the advantages and limitations of emerging sequencing methods, and discuss how the resulting genomic datasets can facilitate precision medicine in clinical care and drug development.
A perspective on C. elegans neurodevelopment: from early visionaries to a booming neuroscience research
Published in Journal of Neurogenetics, 2020
The systematic generation of mutants was a productive conceptual leap for connecting animal and cell physiology to genetic information. Sequencing the C. elegans genome and the subsequently established methodologies for genetic mapping (Wicks, Yeh, Gish, Waterston, & Plasterk, 2001) enabled the identification of all mutations that impair nervous system development. Luckily, albeit unanticipated by Brenner, the external application of double-stranded RNA in C. elegans suppresses gene expression. Needless to say, genome sequencing allows for the recent CRISPR/Cas9 genome editing and reverse genetics by RNA interference (Dickinson & Goldstein, 2016; Kamath et al., 2003). This array of unbiased and targeted gene manipulations allows for comprehensive research of neurodevelopment.