Gene knock-in

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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.

Contents

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]

See also

Related Research Articles

Gene knockouts are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. 

A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. Genetic screens can provide important information on gene function as well as the molecular events that underlie a biological process or pathway. While genome projects have identified an extensive inventory of genes in many different organisms, genetic screens can provide valuable insight as to how those genes function.

<span class="mw-page-title-main">Germline mutation</span> Inherited genetic variation

A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

<span class="mw-page-title-main">Gene targeting</span> Genetic technique that uses homologous recombination to change an endogenous gene

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism. It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombination. Gene targeting can be used to make a range of sizes of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much smaller changes to the existing DNA such as a single base-pair change. Gene targeting relies on the presence of a repair template to introduce the user-defined edits to the DNA. The user will design the repair template to contain the desired edit, flanked by DNA sequence corresponding (homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular genomic region. In this way Gene Targeting is distinct from natural homology-directed repair, during which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA. The alteration of DNA sequence in an organism can be useful in both a research context – for example to understand the biological role of a gene – and in biotechnology, for example to alter the traits of an organism.

Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life. Using the conditional gene knockout technique eliminates many of the side effects from traditional gene knockout. In traditional gene knockout, embryonic death from a gene mutation can occur, and this prevents scientists from studying the gene in adults. Some tissues cannot be studied properly in isolation, so the gene must be inactive in a certain tissue while remaining active in others. With this technology, scientists are able to knockout genes at a specific stage in development and study how the knockout of a gene in one tissue affects the same gene in other tissues.

<span class="mw-page-title-main">Genetically modified mouse</span>

A genetically modified mouse or genetically engineered mouse model (GEMM) is a mouse that has had its genome altered through the use of genetic engineering techniques. Genetically modified mice are commonly used for research or as animal models of human diseases and are also used for research on genes. Together with patient-derived xenografts (PDXs), GEMMs are the most common in vivo models in cancer research. Both approaches are considered complementary and may be used to recapitulate different aspects of disease. GEMMs are also of great interest for drug development, as they facilitate target validation and the study of response, resistance, toxicity and pharmacodynamics.

<span class="mw-page-title-main">Knockout rat</span> Type of genetically engineered rat

A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008.

<span class="mw-page-title-main">Genetically modified animal</span> Animal that has been genetically modified

Genetically modified animals are animals that have been genetically modified for a variety of purposes including producing drugs, enhancing yields, increasing resistance to disease, etc. The vast majority of genetically modified animals are at the research stage while the number close to entering the market remains small.

Isogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro. They are provided with a genetically matched 'normal cell' to provide an isogenic system to research disease biology and novel therapeutic agents. They can be used to model any disease with a genetic foundation. Cancer is one such disease for which isogenic human disease models have been widely used.

The European Conditional Mouse Mutagenesis Program or EUCOMM is an EU-funded program to generate a library of mutant mouse embryonic stem cells for research purposes.

A knockout mouse, or knock-out mouse, is a genetically modified mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or physiology, researchers can infer its probable function.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Mutagenesis (molecular biology technique)</span>

In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.

Breast cancer metastatic mouse models are experimental approaches in which mice are genetically manipulated to develop a mammary tumor leading to distant focal lesions of mammary epithelium created by metastasis. Mammary cancers in mice can be caused by genetic mutations that have been identified in human cancer. This means models can be generated based upon molecular lesions consistent with the human disease.

Perturb-seq refers to a high-throughput method of performing single cell RNA sequencing (scRNA-seq) on pooled genetic perturbation screens. Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics. Perturb-seq is a reverse genetics approach that allows for the investigation of phenotypes at the level of the transcriptome, to elucidate gene functions in many cells, in a massively parallel fashion.

Human germline engineering is the process by which the genome of an individual is edited in such a way that the change is heritable. This is achieved by altering the genes of the germ cells, which then mature into genetically modified eggs and sperm. For safety, ethical, and social reasons, there is broad agreement among the scientific community and the public that germline editing for reproduction is a red line that should not be crossed at this point in time. There are differing public sentiments, however, on whether it may be performed in the future depending on whether the intent would be therapeutic or non-therapeutic.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

References

  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. 1 2 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.
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