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. 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 knock-in.
Gene knock-in has allowed, for the first time, hypothesis-driven studies on gene modifications and resultant phenotypes. Mutations in the human p53 gene, for example, can be induced by exposure to benzo(a)pyrene (BaP) and the mutated copy of the p53 gene can be inserted into mouse genomes. Lung tumors observed in the knock-in mice offer support for the hypothesis of BaP’s carcinogenicity. More recent developments in knock-in 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 knock-in also allows for biallelic modifications to some genes to be generated and the phenotype in mice observed in a single generation, an unprecedented timeframe.