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Concept
Reverse genetics
Summary
Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.
Automated DNA sequencing generates large volumes of genomic sequence data relatively rapidly. Many genetic sequences are discovered in advance of other, less easily obtained, biological information. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism. Reverse genetics systems can also allow the recovery and generation of infectious or defective viruses with desired mutations. This allows the ability to study the virus in vitro and in vivo.
In order to learn the influence a sequence has on phenotype, or to discover its biological function, researchers can engineer a change or disrupt the DNA. After this change has been made a researcher can look for the effect of such alterations in the whole organism. There are several different methods of reverse genetics:
Site-directed mutagenesis is a sophisticated technique that can either change regulatory regions in the promoter of a gene or make subtle codon changes in the open reading frame to identify important amino residues for protein function.
Alternatively, the technique can be used to create null alleles so that the gene is not functional. For example, deletion of a gene by gene targeting (gene knockout) can be done in some organisms, such as yeast, mice and moss. Unique among plants, in Physcomitrella patens, gene knockout via homologous recombination to create knockout moss (see figure) is nearly as efficient as in yeast. In the case of the yeast model system directed deletions have been created in every non-essential gene in the yeast genome.
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Knockout moss
A knockout moss is one kind of genetically modified moss, which are GM plants. One or more of the moss's specific genes are deleted or inactivated ("knocked out"), for example by gene targeting or other methods. After the deletion of a gene, the knockout moss has lost the trait encoded by this gene. Thus, the function of this gene can be inferred. This scientific approach is called reverse genetics as the scientist wants to unravel the function of a specific gene.
Gene knockout
Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs. One of the main advantages of gene knockouts is that they allow researchers to study the function of a specific gene in vivo, and to understand the role of the gene in normal development and physiology as well as in the pathology of diseases.
Zebrafish
The zebrafish (Danio rerio) is a freshwater fish belonging to the minnow family (Cyprinidae) of the order Cypriniformes. Native to India and South Asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio (and thus often called a "tropical fish" although both tropical and subtropical). It is also found in private ponds. The zebrafish is an important and widely used vertebrate model organism in scientific research, for example in drug development, in particular pre-clinical development.
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