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In molecular cloning and biology, a gene knock-in (abbreviation: KI) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion 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 traditional transgenic techniques is that a knock-in involves a gene inserted into a specific locus, and is thus a "targeted" insertion. It is the opposite of gene 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 new phenotype of the organism in question. The BACs and YACs are used in this case so that large fragments can be transferred.

Technique

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 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.^[4]

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.^[5] 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.^[6] 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.^[7]

Versus gene knockout

Knock-in technology is different from knockout technology in that knockout technology aims to either delete part of the DNA sequence or insert 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 a gain-of-function mutation and a gene knockout a loss-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]

Potential applications

Because of the success of gene knock-in methods thus far, many clinical applications can be envisioned. Knock-in of sections of the human immunoglobulin gene into mice has already been shown to allow them to produce humanized antibodies that are therapeutically useful.^[9] It should be possible to modify stem cells in humans to restore targeted gene function in certain tissues, for example possibly correcting the mutant gamma-chain gene of the IL-2 receptor in hematopoietic stem cells to restore lymphocyte development in people with X-linked severe combined immunodeficiency.^[4]

Limitations

While gene knock-in technology has proven to be a powerful technique for the generation of models of human disease and insight into proteins in 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-explain phenotypes. Also, only a few loci, such as the ROSA26 locus have been characterized well enough where they can be used for conditional gene knock-ins; making combinations of reporter 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 human orthologs 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 the ΔF508 fibrosis mutation in the CFTR gene, which accounts for more than 70% of the mutations in this gene for the human population and leads to cystic 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]

References

^ Gibson, Greg (2009). A Primer Of Genome Science 3rd ed. Sunderland, Massachusetts: Sinauer. pp. 301–302. ISBN 978-0-87893-236-8.
^ Mouse Genome Sequencing Consortium; Waterston, Robert H.; Lindblad-Toh, Kerstin; Birney, Ewan; Rogers, Jane; Abril, Josep F.; Agarwal, Pankaj; Agarwala, Richa; Ainscough, Rachel (2002-12-05). "Initial sequencing and comparative analysis of the mouse genome". Nature. 420 (6915): 520–562. Bibcode:2002Natur.420..520W. doi:10.1038/nature01262. ISSN 0028-0836. PMID 12466850.
^ Westphal, C. H.; Leder, P. (1997-07-01). "Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice". Current Biology. 7 (7): 530–533. Bibcode:1997CBio....7..530W. doi:10.1016/s0960-9822(06)00224-7. ISSN 0960-9822. PMID 9210379.
^ ^a ^b Manis, John P. (2007-12-13). "Knock out, knock in, knock down--genetically manipulated mice and the Nobel Prize". The New England Journal of Medicine. 357 (24): 2426–2429. doi:10.1056/NEJMp0707712. ISSN 1533-4406. PMID 18077807.
^ Liu, Zhipei; Muehlbauer, Karl-Rudolf; Schmeiser, Heinz H.; Hergenhahn, Manfred; Belharazem, Djeda; Hollstein, Monica C. (2005-04-01). "p53 mutations in benzo(a)pyrene-exposed human p53 knock-in murine fibroblasts correlate with p53 mutations in human lung tumors". Cancer Research. 65 (7): 2583–2587. doi:10.1158/0008-5472.CAN-04-3675. ISSN 0008-5472. PMID 15805253.
^ Ruan, Jinxue; Li, Hegang; Xu, Kui; Wu, Tianwen; Wei, Jingliang; Zhou, Rong; Liu, Zhiguo; Mu, Yulian; Yang, Shulin (2015-09-18). "Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs". Scientific Reports. 5: 14253. Bibcode:2015NatSR...514253R. doi:10.1038/srep14253. PMC 4585612. PMID 26381350.
^ Wang, Yanliang; Li, Junhong; Xiang, Jinzhu; Wen, Bingqiang; Mu, Haiyuan; Zhang, Wei; Han, Jianyong (2015-12-10). "Highly efficient generation of biallelic reporter gene knock-in mice via CRISPR-mediated genome editing of ESCs". Protein & Cell. 7 (2): 152–156. doi:10.1007/s13238-015-0228-3. ISSN 1674-800X. PMC 4742388. PMID 26661644.
^ Doyle, Alfred; McGarry, Michael P.; Lee, Nancy A.; Lee, James J. (2012-04-01). "The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease". Transgenic Research. 21 (2): 327–349. doi:10.1007/s11248-011-9537-3. ISSN 0962-8819. PMC 3516403. PMID 21800101.
^ Benatuil, Lorenzo; Kaye, Joel; Cretin, Nathalie; Godwin, Jonathan G.; Cariappa, Annaiah; Pillai, Shiv; Iacomini, John (2008-03-15). "Ig knock-in mice producing anti-carbohydrate antibodies: breakthrough of B cells producing low affinity anti-self antibodies". Journal of Immunology. 180 (6): 3839–3848. doi:10.4049/jimmunol.180.6.3839. ISSN 0022-1767. PMID 18322191.
^ Tellkamp, Frederik; Benhadou, Farida; Bremer, Jeroen; Gnarra, Maria; Knüver, Jana; Schaffenrath, Sandra; Vorhagen, Susanne (2014-12-01). "Transgenic mouse technology in skin biology: generation of knockin mice". The Journal of Investigative Dermatology. 134 (12): 1–3. doi:10.1038/jid.2014.434. ISSN 1523-1747. PMID 25381772.
^ Grubb, Barbara R.; Boucher, Richard C. (1999-01-01). "Pathophysiology of Gene-Targeted Mouse Models for Cystic Fibrosis". Physiological Reviews. 79 (1): S193 – S214. doi:10.1152/physrev.1999.79.1.S193. ISSN 0031-9333. PMID 9922382.
^ Rogers, Christopher S.; Hao, Yanhong; Rokhlina, Tatiana; Samuel, Melissa; Stoltz, David A.; Li, Yuhong; Petroff, Elena; Vermeer, Daniel W.; Kabel, Amanda C. (2008-04-01). "Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer". The Journal of Clinical Investigation. 118 (4): 1571–1577. doi:10.1172/JCI34773. ISSN 0021-9738. PMC 2265103. PMID 18324337.