Inmolecular cloning andbiology, agene knock-in (abbreviation:KI) refers to agenetic engineering method that involves the one-for-one substitution of DNA sequence information in agenetic locus or theinsertion of sequence information not found within the locus.[1] 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.[2] The difference between knock-in technology and traditionaltransgenic techniques is that a knock-in involves agene inserted into a specific locus, and is thus a "targeted" insertion. It is the opposite ofgene knockout.
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 newphenotype of the organism in question. TheBACs andYACs are used in this case so that large fragments can be transferred.
Gene knock-in originated as a slight modification of the original knockout technique developed byMartin Evans,Oliver Smithies, andMario Capecchi. Traditionally, knock-in techniques have relied onhomologous recombination to drive targeted gene replacement, although other methods using atransposon-mediated system to insert the target gene have been developed.[3] The use ofloxP flanking sites that become excised upon expression ofCre recombinase with gene vectors is an example of this.Embryonic stem cells with the modification of interest are then implanted into a viableblastocyst, which will grow into a maturechimeric 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.[4]
Gene knock-in has allowed, for the first time, hypothesis-driven studies on gene modifications and resultant phenotypes. Mutations in the humanp53 gene, for example, can be induced by exposure tobenzo(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’scarcinogenicity.[5] More recent developments in knock-in technique have allowed for pigs to have a gene forgreen fluorescent protein inserted with aCRISPR/Cas9 system, which allows for much more accurate and successful gene insertions.[6] The speed of CRISPR/Cas9-mediated gene knock-in also allows forbiallelic modifications to some genes to be generated and the phenotype in mice observed in a single generation, an unprecedented timeframe.[7]
Knock-in technology is different fromknockout technology in that knockout technology aims to eitherdelete part of the DNA sequence orinsert irrelevant DNA sequence information to disrupt the expression of a specific genetic locus. Gene knock-in technology, on the other hand, alters the genetic locus of interest via a one-for-one substitution of DNA sequence information or by the addition of sequence information that is not found on said genetic locus. A gene knock-in therefore can be seen as again-of-function mutation and a gene knockout aloss-of-function mutation, but a gene knock-in may also involve the substitution of a functional gene locus for a mutant phenotype that results in some loss of function.[8]
Because of the success of gene knock-in methods thus far, many clinical applications can be envisioned. Knock-in of sections of the humanimmunoglobulin gene into mice has already been shown to allow them to produce humanized antibodies that are therapeutically useful.[9] It should be possible to modifystem cells in humans to restore targeted gene function in certain tissues, for example possibly correcting the mutant gamma-chain gene of theIL-2 receptor inhematopoietic stem cells to restorelymphocyte development in people with X-linkedsevere combined immunodeficiency.[4]
While gene knock-in technology has proven to be a powerful technique for the generation of models of human disease and insight into proteinsin vivo, numerous limitations still exist. Many of these are shared with the limitations of knockout technology. First, combinations of knock-in genes lead to growing complexity in the interactions that inserted genes and their products have with other sections of the genome and can therefore lead to more side effects and difficult-to-explainphenotypes. Also, only a few loci, such as theROSA26 locus have been characterized well enough where they can be used for conditional gene knock-ins; making combinations ofreporter and transgenes in the same locus problematic. The biggest disadvantage of using gene knock-in for human disease model generation is that mouse physiology is not identical to that of humans and humanorthologs of proteins expressed in mice will often not wholly reflect the role of a gene in human pathology.[10] This can be seen in mice produced with theΔF508 fibrosis mutation in theCFTR gene, which accounts for more than 70% of the mutations in this gene for the human population and leads tocystic fibrosis. While ΔF508 CF mice do exhibit the processing defects characteristic of the human mutation, they do not display the pulmonary pathophysiological changes seen in humans and carry virtually no lung phenotype.[11] Such problems could be ameliorated by the use of a variety of animal models, and pig models (pig lungs share many biochemical and physiological similarities with human lungs) have been generated in an attempt to better explain the activity of the ΔF508 mutation.[12]