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Gene knock-in

April 10, 2024

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 edit

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 edit

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 edit

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 edit

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]

See also edit

References edit

  1. ^ Gibson, Greg (2009). A Primer Of Genome Science 3rd ed. Sunderland, Massachusetts: Sinauer. pp. 301–302. ISBN 978-0-87893-236-8.
  2. ^ 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.
  3. ^ 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. doi:10.1016/s0960-9822(06)00224-7. ISSN 0960-9822. PMID 9210379.
  4. ^ 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.
  5. ^ 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.
  6. ^ 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.
  7. ^ 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.
  8. ^ 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.
  9. ^ 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.
  10. ^ 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.
  11. ^ 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.
  12. ^ 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.

External links edit

April 10, 2024
gene, knock, molecular, cloning, biology, gene, knock, abbreviation, refers, genetic, engineering, method, that, involves, substitution, sequence, information, genetic, locus, insertion, sequence, information, found, within, locus, typically, this, done, mice,. 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 Contents 1 Technique 2 Versus gene knockout 3 Potential applications 4 Limitations 5 See also 6 References 7 External linksTechnique editGene 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 editKnock 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 editBecause 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 editWhile 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 DF508 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 DF508 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 DF508 mutation 12 See also editGene knockout Genetic engineering Genetic recombination Molecular cloning Plasmid Vector molecular biology References edit 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 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 amp 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 Knuver 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 External links editGenetic methods techniques and protocols Koch Institute for Integrative Cancer Research at MIT Knockins and Knockouts UMass Profiles Research Networking Software Gene Knock In Techniques a research networking and expertise mining software tool http www transgenic co jp en products mice service modified mouse knockin php outlines the process of constructing insertion vectors and breeding mice Retrieved from https en wikipedia org w index php title Gene knock in amp oldid 1192814081, wikipedia, wiki, book, books, library,

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