Reverse genetics
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Reverse genetics is an approach to discovering the function of a gene that proceeds in the opposite direction of so called forward genetic screens that are more usual in classical genetics.
Classical and reverse genetics are alike since using either approach investigators try to deduce information from the effects of damaging or changing a gene's function. By the classical approach, geneticists first look for rare individuals with an unusual phenotype and then attempt to identify the unknown faulty allele or gene. In contrast, for reverse genetics, the goal is to identify a known gene's phenotype.
Due to the modern techniques of DNA sequencing, vast amounts of genomic sequence data become available and many genetic sequences are discovered in advance of other information. 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 approaches to identify new alleles in a known gene.
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Random deletions, insertions and point mutations
These are three similar techniques that involve creating large mutagenised populations in a similar way to forward genetic screens. These populations are generated using either chemical (point mutations), gamma radiation (deletions) or DNA insertions (insertional knockouts). These large libraries of mutants can be screened for specific changes at the gene of interest using PCR. For some organisms, such as Drosophila and Arabidopsis there are large online databases that indicate the locations of all the DNA insertions in a particular library.
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 knockout can be done in some organisms, such as yeast and mice. In the case of the yeast model system directed deletions have been created in every non-essential gene in the yeast genome.
In some cases conditional alleles can be used that have normal function until the allele is activated. This is known as gene knockin. 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 specfic 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.
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.
| Subfields of genetics |
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| Classical genetics | Ecological genetics | Molecular genetics | Population genetics | Quantitative genetics |
| Related topics: Genomics | Reverse genetics | Geneticist |