Gene knockin

In molecular cloning and biology, a knock-in (or gene knock-in) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information with a wild-type copy in a genetic locus or the insertion of sequence information not found within the locus. Typically, this is done in mice since the technology for this process is more refined and there is a high degree of shared sequence complexity between mice and humans. The difference between knock-in technology and traditional transgenic techniques is that a knock-in involves a gene inserted into a specific locus, and is thus a "targeted" insertion.

A common use of knock-in technology is for the creation of disease models. It is a technique by which scientific investigators may study the function of the regulatory machinery (e.g. promoters) that governs the expression of the natural gene being replaced. This is accomplished by observing the new phenotype of the organism in question. The BACs and YACs are used in this case so that large fragments can be transferred.

Gene knockin originated as a slight modification of the original knockout technique developed by Martin Evans, Oliver Smithies, and Mario Capecchi. Traditionally, knockin 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. The use of loxP flanking sites that become excised upon expression of Cre recombinase with gene vectors is an example of this. Embryonic stem cells with the modification of interest are then implanted into a viable blastocyst, which will grow into a mature chimeric mouse with some cells having the original blastocyst cell genetic information and other cells having the modifications introduced to the embryonic stem cells. Subsequent offspring of the chimeric mouse will then have the gene knockin.

Gene knockin has allowed, for the first time, hypothesis-driven studies on gene modifications and resultant phenotypes. Mutations in the human p53 gene, for example, can induced by exposure to benzo(a)pyrene and the mutated copy of the p53 gene can be inserted into mouse genomes. Lung tumors observed in the knockin mice offer support for the hypothesis of BaP’s carcinogenicity. More recent developments in knockin technique have allowed for pigs to have a gene for green fluorescent protein inserted with a CRISPR/Cas9 system, which allows for much more accurate and successful gene insertions. The speed of CRISPR/Cas9-mediated gene knockin also allows for biallelic modifications to some genes to be generated and the phenotype in mice observed in a single generation, an unprecedented timeframe.

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