Gene Knockout 2022 20th March 2022 – Tags: gene, gene knockout
Gene knockout, also called gene inactivation, is a genetic technique in which the expression of a particular gene in an organism is suppressed by replacing the original gene at its genetic locus with a modified version of the gene in which one or more exons have been deleted, resulting in organisms that do not express the gene in a particular tissue or in the whole organism. These organisms are also called knockouts.
A knockout is identical to the above, but instead of deleting a particular gene, it replaces it with an altered version of the gene that produces an additional function.
gene knockout overview
Gene blocking technologies are used to alter the genomes of all living organisms. When a mutation inactivates the function of a gene, it is called a gene knockout. Gene knockout methods are used to identify the function of a particular gene by inhibiting the function of that gene.
This process is used in both classical genetics and modern techniques such as functional genomics. Initially, gene knockout was performed by transposon mutagenesis. The main disadvantage of this method is the tedious screening to find the neutralised gene. Deletion of other organisms was achieved with the help of genetic engineering.
In vitro techniques are used to modify genes in plasmids or bacterial artificial chromosomes (BACs), and these modified constructs are then transferred to the organism in question using cell culture techniques. Other methods use a combination of genetic engineering and homologous recombination in vivo. However, this combination is not as effective.
Homologous recombination is the ultimate tool for producing gene knockouts. It allows direct declaration of the bacterial chromosome or modification of any plasmid or BAC in vivo as precursors. This technique does not rely on restriction sites. A drug cassette can be placed anywhere in a gene or the open reading of the gene can be replaced by the drug cassette. In either case, the desired construct is selected.
Gene knockout Method
Different molecular biology techniques are available to influence gene expression. These techniques differ in the instruments used, the time required to obtain a positive result and the specificity of the result. These techniques include:
- Homologous recombination: the original gene is replaced by a mutated version. This mutated version can eliminate almost the entire gene sequence or leave the expression of the first exons. It usually takes several months to obtain modified individuals (especially in mice), although they have the advantage of being very specific: these organisms lack only the target gene.
- Transgénesis, which consists of introducing sequences into the original gene that modify it to produce changes that result in non-functional proteins. This is a generally faster technique, the disadvantage of which is that the endogenous gene is still present and can produce a protein, even a mutated one, which can affect other molecules in the metabolic pathway to which it belongs. In addition, a new mutation can occur by chance and reverse the introduced modification by producing the original protein again, which is not uncommon.
- Mutagenesis. Mutants can be obtained using a mutagenic product (e.g. EMS) that produces random mutations. Organisms carrying “STOP” codons in the desired sequence are then selected. This is a rapid method for obtaining mutants, although it is not specific for the target gene, which can lead to setbacks.
- Demolition. Las secuDNA enzymes expressing interfering RNA molecules can be introduced into the genome of an organism or directly into cells. These molecules specifically interfere with the expression of the target gene, usually resulting in reduced expression (for this reason they are also called “knockdown”). This technique is faster than obtaining a knock-out organism, but does not result in complete elimination of expression, and the introduced sequence encoding the interfering RNA may also undergo mutations that inactivate the desired effect.
Genetic knockout technologies
The best approach to produce a gene knockout is homologous recombination. Using gene knockout methods, a single gene is eliminated without affecting all other genes in an organism. With this method, the organism in which the gene in question is no longer functional is called a knockout organism. When more than one gene is deleted in an organism, it is called a double knock-out or DKO, triple knock-out or TKO and quadruple knock-out or QKO, depending on the number of genes.
Genetic engineering methods
Gene knockout is carried out with elements such as plasmids, DNA constructs or artificial bacterial chromosomes.
The gene knock-out process usually produces transgenic animals in which the target gene has been altered. To create transgenic animals, embryonic stem or ES cells are genetically modified and in the next step the transformed ES cells are inserted into early embryos.
The transformed animals produced in this way have the ability to pass on the transformed gene to subsequent generations.
gene knockout theory
Recombination is defined as genetic engineering mediated by homologous recombination in vivo. Recombination is performed with the help of a bacteriophage. Bacteriophages such as λ Red, RecET or similar systems are most commonly used. Recombination can also be used for deletions, point mutations, duplications, inversions, fusions and tags. A linear DNA substrate containing the desired modification or homologies is introduced into the target DNA of the cells. The cells express recombinant enzymes encoded by the phage. These enzymes help to incorporate the linear DNA into the target molecule. In this way, recombinant molecules are produced. The most commonly used recombination system is that of the bacteriophage λ Red. This system consists of the three Gam proteins, Exo Beta. The Gam protein inhibits the E. coli RecBCD exonuclease, which normally degrades linear dsDNA. Gam is not essential for recombination, but increases the recombination frequency of dsDNA up to 20-fold. Exo is an exonuclease specific for 5’→3′ double-stranded DNA and is required for dsDNA recombination. Protein beta, a DNA annexin protein, is the central recombinase for recombination.
Importantly, host recombination functions, including the key recombination protein RecA, are not required for recombination.