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Reverse genetics is a method in
molecular genetics Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the ...
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 Classical genetics is the branch of genetics based solely on visible results of reproductive acts. It is the oldest discipline in the field of genetics, going back to the experiments on Mendelian inheritance by Gregor Mendel who made it possible t ...
. 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''.


Techniques used

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:


Directed deletions and point mutations

Site-directed mutagenesis Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...
is a sophisticated technique that can either change regulatory regions in the promoter of a gene or make subtle
codon The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links p ...
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 targeting (also, replacement strategy based on homologous recombination) is a genetic technique that uses homologous recombination to modify an endogenous gene. The method can be used to delete a gene, remove exons, add a gene and modify ...
( gene knockout) can be done in some organisms, such as yeast, mice and moss. Unique among plants, in ''
Physcomitrella patens ''Physcomitrium patens'', (synonym: ''Physcomitrella patens'' ) the spreading earthmoss, is a moss (bryophyte) used as a model organism for studies on plant evolution, development, and physiology. Distribution and ecology ''Physcomitrella p ...
'', gene knockout via
homologous recombination Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may be ...
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. In the case of the plant model system huge mutant libraries have been created based on gene disruption constructs. In
gene knock-in In molecular cloning and biology, a gene knock-in (abbreviation: KI) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found ...
, 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 point mutations in a target gene. In the field of virology, reverse-genetics techniques can be used to recover full-length infectious viruses with desired mutations or insertions in the viral genomes or in specific virus genes. Technologies that allow these manipulations include circular polymerase extension reaction (CPER) which was first used to generate infectious cDNA for Kunjin virus a close relative of West Nile virus. CPER has also been successfully utilised to generate a range of positive-sense RNA viruses such as SARS-CoV-2, the causative agent of COVID-19.


Gene silencing

The discovery of
gene silencing Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence gen ...
using double stranded RNA, also known as RNA interference (RNAi), and the development of gene knockdown using
Morpholino A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
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 is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleot ...
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 ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...
'', 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). RNAi interference, specifically gene silencing, has become a useful tool to silence the expression of genes and identify and analyze their loss-of-function phenotype. When mutations occur in alleles, the function which it represents and encodes also is mutated and lost; this is generally called a loss-of-function mutation. The ability to analyze the loss-of-function phenotype allows analysis of gene function when there is no access to mutant alleles. 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 A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
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 Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the ...
approach is the creation of
transgenic A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
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 The wild type (WT) is 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, "m ...
) gene's function. For example, over-expression of a mutant gene may result in high levels of a non-functional protein resulting in a
dominant negative In genetics, dominance is the phenomenon of one variant (allele) of a gene on a chromosome masking or overriding the effect of a different variant of the same gene on the other copy of the chromosome. The first variant is termed dominant and ...
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 In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
, or ubiquitination). Either change is critical for modulating protein function and often result in informative phenotypes.


Vaccine synthesis

Reverse genetics plays a large role in vaccine synthesis. Vaccines can be created by engineering novel genotypes of infectious viral strains which diminish their pathogenic potency enough to facilitate immunity in a host. The reverse genetics approach to vaccine synthesis utilizes known viral genetic sequences to create a desired phenotype: a virus with both a weakened pathological potency and a similarity to the current circulating virus strain. Reverse genetics provides a convenient alternative to the traditional method of creating inactivated vaccines, viruses which have been killed using heat or other chemical methods. Vaccines created through reverse genetics methods are known as
attenuated vaccine An attenuated vaccine (or a live attenuated vaccine, LAV) is a vaccine created by reducing the virulence of a pathogen, but still keeping it viable (or "live"). Attenuation takes an infectious agent and alters it so that it becomes harmless or les ...
s, named because they contain weakened (attenuated) live viruses. Attenuated vaccines are created by combining genes from a novel or current virus strain with previously attenuated viruses of the same species. Attenuated viruses are created by propagating a live virus under novel conditions, such as a chicken's egg. This produces a viral strain that is still live, but not pathogenic to humans, as these viruses are rendered defective in that they cannot replicate their genome enough to propagate and sufficiently infect a host. However, the viral genes are still expressed in the host's cell through a single replication cycle, allowing for the development of an immunity.


Influenza vaccine

A common way to create a vaccine using reverse genetic techniques is to utilize plasmids to synthesize attenuated viruses. This technique is most commonly used in the yearly production of
influenza vaccine Influenza vaccines, also known as flu shots, are vaccines that protect against infection by influenza viruses. New versions of the vaccines are developed twice a year, as the influenza virus rapidly changes. While their effectiveness varies fro ...
s, where an eight plasmid system can rapidly produce an effective vaccine. The entire genome of the influenza A virus consists of eight RNA. segments, so the combination of six attenuated viral
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
plasmids with two wild-type plasmids allow for an attenuated vaccine strain to be constructed. For the development of influenza vaccines, the fourth and sixth RNA segments, encoding for the hemagglutinin and neuraminidase proteins respectively, are taken from the circulating virus, while the other six segments are derived from a previously attenuated master strain. The HA and NA proteins exhibit high
antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
variety, and therefore are taken from the current strain for which the vaccine is being produced to create a well matching vaccine. The plasmid used in this eight-plasmid system contains three major components that allow for vaccine development. Firstly, the plasmid contains
restriction sites Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (beca ...
that will enable the incorporation of influenza genes into the plasmid. Secondly, the plasmid contains an antibiotic resistance gene, allowing the selection of merely plasmids containing the correct gene. Lastly, the plasmid contains two promotors, human pol 1 and pol 2 promotor that transcribe genes in opposite directions. cDNA sequences of viral RNA are synthesized from attenuated master strains by using
RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chai ...
. This cDNA can then be inserted between an RNA polymerase I (Pol I) promoter and terminator sequence through restriction enzyme digestion. The cDNA and pol I sequence is then, in turn, surrounded by an RNA polymerase II (Pol II) promoter and a polyadenylation site. This entire sequence is then inserted into a plasmid. Six plasmids derived from attenuated master strain cDNA are cotransfected into a target cell, often a chicken egg, alongside two plasmids of the currently circulating wild-type influenza strain. Inside the target cell, the two "stacked" Pol I and Pol II enzymes transcribe the viral cDNA to synthesize both negative-sense viral RNA and positive-sense mRNA, effectively creating an attenuated virus. The result is a defective vaccine strain that is similar to the current virus strain, allowing a host to build immunity. This synthesized vaccine strain can then be used as a seed virus to create further vaccines.


Advantages and disadvantages

Vaccines engineered from reverse genetics carry several advantages over traditional vaccine designs. Most notable is speed of production. Due to the high antigenic variation in the HA and NA glycoproteins, a reverse-genetic approach allows for the necessary genotype (i.e. one containing HA and NA proteins taken from currently circulating virus strains) to be formulated rapidly. Additionally, since the final product of a reverse genetics attenuated vaccine production is a live virus, a higher immunogenicity is exhibited than in traditional inactivated vaccines, which must be killed using chemical procedures before being transferred as a vaccine. However, due to the live nature of attenuated viruses, complications may arise in
immunodeficient Immunodeficiency, also known as immunocompromisation, is a state in which the immune system's ability to fight infectious diseases and cancer is compromised or entirely absent. Most cases are acquired ("secondary") due to extrinsic factors that a ...
patients. There is also the possibility that a mutation in the virus could result the vaccine to turning back into a live unattenuated virus.


See also

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Forward genetics Forward genetics is a molecular genetics approach of determining the genetic basis responsible for a phenotype. Forward genetics provides an unbiased approach because it relies heavily on identifying the genes or genetic factors that cause a partic ...


References


Further reading

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External links


Reassortment vs. Reverse Genetics

Reverse Genetics: Building Flu Vaccines Piece by Piece
{{DEFAULTSORT:Reverse Genetics Genetic engineering Molecular genetics