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Gene knockout by mutation is commonly carried out in bacteria. An early instance of the use of this technique in Escherichia coli was published in 1989 by Hamilton, et al. [2] In this experiment, two sequential recombinations were used to delete the gene.
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 oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
Floxing a gene allows it to be deleted (knocked out), [5] [6] translocated or inserted [7] (through various mechanisms in Cre-Lox recombination). The floxing of genes is essential in the development of scientific model systems as it allows spatial and temporal alteration of gene expression.
In addition, it has been used to engineer stably modified human embryonic stem cell and induced pluripotent stem cell (IPSCs) clones and human erythroid cell lines, [11] [28] to generate knockout C. elegans, [12] knockout rats, [13] knockout mice, [29] and knockout zebrafish. [14] [30] Moreover, the method can be used to generate knockin organisms.
Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. [1] [2] This technique is useful to study the role of individual genes in living organisms.
Vector containing DNA sequence similar to the gene to be modified is introduced to the cell, and by a process of recombination replaces the target gene in the chromosome. This method can be used to introduce a mutation or knock out a gene, for example as used in the production of knockout mice. [29]
Gene knock-in originated as a slight modification of the original knockout technique developed by Martin Evans, Oliver Smithies, and Mario Capecchi.Traditionally, knock-in techniques have relied on homologous recombination to drive targeted gene replacement, although other methods using a transposon-mediated system to insert the target gene have been developed. [3]
The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and ...