During the last decade, the term reverse genetics has been frequently used for physical mapping and isolation of genes whose protein products are unknown. This has been suggested in contrast to forward genetics, where genes are mapped on the basis of phenotype, using the techniques of classical genetics. Recently (in 1991) the meaning of the term reverse genetics has been redefined. It has been argued that in forward genetics, we start the study on the basis of phenotype, leading ultimately to the study of DNA sequences comprising the gene for this phenotype. Therefore, even though the mapping and isolation of a gene for any trait (including those for human diseases) with unknown gene product is a spectacular achievement, but the approach cannot be termed reverse genetics for the simple reason that in this case also, we start the genetic study on the basis of phenotype and finally isolate the gene, albeit using sophisticated techniques of recombinant DNA. Therefore, Paul Berg (Nobel Laureate) in a recent report (1991) suggested that the usage of the term reverse genetics be restricted to those studies, where we start the study with a DNA segment with unknown phenotypic effect, introduce this DNA (without any alteration or after modification) into a plant or an animal and then study its phenotypic effect. Production of transgenic plants and animals followed by a study of their phenotype or identification of regulatory DNA sequences using transgenic plants or animals or targeted alterations in genes at the molecular level are some examples of reverse genetics. These techniques of reverse genetics will be increasingly used in future leading to significant advances in our knowledge of genetics.
Reverse genetics is an approach to discover the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing. This investigative process proceeds in the opposite direction of so-called forward genetic screens of classical genetics. Simply put, while forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes arise as a result of particular genes.
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.
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Techniques used in reverse genetics
To learn the influence a sequence has on phenotype, or to discover its biological function, researchers can engineer a change or disruption in 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 that have proved useful:
Directed deletions and point mutations
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 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. In the case of the plant model system huge mutant libraries have been created based on gene disruption constructs. In gene knock-in, the endogenous exon is replaced by an altered sequence of interest.
In some cases conditional alleles can be used so that the gene has normal function until the conditional allele is activated. This might entail ‘knocking in’ recombinase sites (such as lox or frt sites) that will cause a deletion at the gene of interest when a specific recombinase (such as CRE, FLP) is induced. Cre or Flp recombinases can be induced with chemical treatments, heat shock treatments or be restricted to a specific subset of tissues.
Another technique that can be used is TILLING . This is a method that combines a standard and efficient technique of mutagenesis with a chemical mutagen such as Ethyl methanesulfonate (EMS) with a sensitive DNA screening-technique that identifies single base mutations (also called point mutations) in a target gene.
Gene silencing
The discovery of gene silencing using double stranded RNA, also known as RNA interference (RNAi), and the development of gene knockdown using Morpholino oligos, have made disrupting gene expression an accessible technique for many more investigators. This method is often referred to as a gene knockdown since the effects of these reagents are generally temporary, in contrast to gene knockouts which are permanent.
RNAi creates a specific knockout effect without actually mutating the DNA of interest. In C. elegans, RNAi has been used to systematically interfere with the expression of most genes in the genome. RNAi acts by directing cellular systems to degrade target messenger RNA (mRNA).
While RNA interference relies on cellular components for efficacy (e.g. the Dicer proteins, the RISC complex) a simple alternative for gene knockdown is Morpholino antisense oligos. Morpholinos bind and block access to the target mRNA without requiring the activity of cellular proteins and without necessarily accelerating mRNA degradation. Morpholinos are effective in systems ranging in complexity from cell-free translation in a test tube to in vivo studies in large animal models.
Interference using transgenes
A molecular genetic approach is the creation of transgenic organisms that overexpress a normal gene of interest. The resulting phenotype may reflect the normal function of the gene.
Alternatively it is possible to overexpress mutant forms of a gene that interfere with the normal (wildtype) genes function. For example, over expression of a mutant gene may result in high levels of a non-functional protein resulting in a dominant negative interaction with the wildtype protein. In this case the mutant version will out compete for the wildtype proteins partners resulting in a mutant phenotype.
Other mutant forms can result in a protein that is abnormally regulated and constitutively active (‘on’ all the time). This might be due to removing a regulatory domain or mutating a specific amino residue that is reversibly modified (by phosphorylation methylation or ubiquitination). Either change is critical for modulating protein function and often result in informative phenotypes.