Reverse genetics
Encyclopedia
Reverse genetics is an approach to discovering 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.
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 allele
s 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 (see TILLING (molecular biology)
). 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.
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 knockout
s 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.
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 ubiquitin
ation). Either change is critical for modulating protein function and often result in informative phenotypes.
}
}
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Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
. This investigative process proceeds in the opposite direction of so-called forward genetic
Forward genetics
Forward genetics is an approach that encompasses several means of identifying the gene or set of genes that are responsible for a particular phenotype within an organism. Initially, this entailed the generation of random mutations in an organism, often through radiation or insertional mutagenesis...
screens of classical genetics
Classical genetics
Classical genetics consists of the technique and methodologies of genetics that predate the advent of molecular biology. A key discovery of classical genetics in eukaryotes was genetic linkage...
. Simply put, while forward genetics seeks to find the genetic basis of a phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
or trait, reverse genetics seeks to find what phenotypes arise as a result of particular genes.
Automated DNA sequencing
Sequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
generates large volumes of genomic
Genomics
Genomics is a discipline in genetics concerning the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis,...
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.
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 mutagenesisSite-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
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
Open reading frame
In molecular genetics, an open reading frame is a DNA sequence that does not contain a stop codon in a given reading frame.Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop...
to identify important amino residues for protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
function.
Alternatively, the technique can be used to create null allele
Null allele
A null allele is a mutant copy of a gene that completely lacks that gene's normal function. This can be the result of the complete absence of the gene product at the molecular level, or the expression of a non-functional gene product...
s so that the gene is not functional. For example, deletion of a gene by gene targeting
Gene targeting
Gene targeting is a genetic technique that uses homologous recombination to change an endogenous gene. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Gene targeting can be permanent or conditional...
(gene knockout
Gene knockout
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
) can be done in some organisms, such as yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
, mice
Knockout mouse
A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA...
and moss
Moss
Mosses are small, soft plants that are typically 1–10 cm tall, though some species are much larger. They commonly grow close together in clumps or mats in damp or shady locations. They do not have flowers or seeds, and their simple leaves cover the thin wiry stems...
. Unique among plants, in Physcomitrella patens
Physcomitrella patens
Physcomitrella patens is a moss used as a model organism for studies on plant evolution, development and physiology.-Model organism:...
, gene knockout via homologous recombination
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
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
Scientific modelling
Scientific modelling is the process of generating abstract, conceptual, graphical and/or mathematical models. Science offers a growing collection of methods, techniques and theory about all kinds of specialized scientific modelling...
huge mutant libraries have been created based on gene disruption constructs. In gene knock-in
Gene knockin
In molecular cloning and biology, a Knock-in refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome. Typically, this is done in mice since the technology for this process is more refined, and because...
, 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 (see TILLING (molecular biology)
Tilling (molecular biology)
TILLING is a method in molecular biology that allows directed identification of mutations in a specific gene. TILLING was introduced in 2000, using the model plant Arabidopsis thaliana...
). 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 silencingGene silencing
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification...
using double stranded RNA, also known as RNA interference
RNA interference
RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...
(RNAi), and the development of gene knockdown using Morpholino
Morpholino
In molecular biology, a Morpholino is a molecule in a particular structural family that is used to modify gene expression. Morpholino oligomers are an antisense technology used to block access of other molecules to specific sequences within nucleic acid...
oligos, have made disrupting gene expression an accessible technique for many more investigators. This method is often referred to as a gene knockdown
Gene knockdown
Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene...
since the effects of these reagents are generally temporary, in contrast to gene knockout
Gene knockout
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
s which are permanent.
RNAi creates a specific knockout effect without actually mutating the DNA of interest. In C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, 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
RNA interference
RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...
relies on cellular components for efficacy (e.g. the Dicer proteins, the RISC complex) a simple alternative for gene knockdown is Morpholino
Morpholino
In molecular biology, a Morpholino is a molecule in a particular structural family that is used to modify gene expression. Morpholino oligomers are an antisense technology used to block access of other molecules to specific sequences within nucleic acid...
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
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
studies in large animal models.
Interference using transgenes
A molecular geneticMolecular genetics
Molecular genetics is the field of biology and genetics that studies the structure and function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology...
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
Wild type
Wild type refers to the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard, "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele...
) 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
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
or ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
ation). Either change is critical for modulating protein function and often result in informative phenotypes.
External links
- From the http://www3.niaid.nih.gov/National Institute of Allergy and Infectious DiseasesNational Institute of Allergy and Infectious DiseasesThe National Institute of Allergy and Infectious Diseases is one of the 27 institutes and centers that make up the National Institutes of Health , an agency of the United States Department of Health and Human Services...
] (NIAID) site: - From the http://www.ncbi.nlm.nih.gov/National Center for Biotechnology InformationThe National Center for Biotechnology Information is part of the United States National Library of Medicine , a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper...
(NCBI) site]:
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