Conditional gene knockout

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Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. [1] [2] 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. [3] [4]

Contents

Technique

Diagram showing how to generate a conditional knockout mouse: A mouse containing the Cre gene and a mouse containing the lox gene were bred to generate a conditional knockout for a particular gene of interest. The mice do not naturally express Cre recombinase or lox sites, but they have been engineered to express these gene products to create the desirable offspring. Conditional Knockout Mouse.jpg
Diagram showing how to generate a conditional knockout mouse: A mouse containing the Cre gene and a mouse containing the lox gene were bred to generate a conditional knockout for a particular gene of interest. The mice do not naturally express Cre recombinase or lox sites, but they have been engineered to express these gene products to create the desirable offspring.

The most commonly used technique is the Cre-lox recombination system. The Cre recombinase enzyme specifically recognizes two lox (loci of recombination) sites within DNA and causes recombination between them. During recombination two strands of DNA exchange information. This recombination will cause a deletion or inversion of the genes between the two lox sites, depending on their orientation. An entire gene can be removed to inactivate it. [1] [3] This whole system is inducible so a chemical can be added to knock genes out at a specific time. Two of the most commonly used chemicals are tetracycline, which activates transcription of the Cre recombinase gene and tamoxifen, which activates transport of the Cre recombinase protein to the nucleus. [4] Only a few cell types express Cre recombinase and no mammalian cells express it so there is no risk of accidental activation of lox sites when using conditional gene knockout in mammals. Figuring out how to express Cre-recombinase in an organism tends to be the most difficult part of this technique. [3]

Uses

The conditional gene knockout method is often used to model human diseases in other mammals. [2] It has increased scientists’ ability to study diseases, such as cancer, that develop in specific cell types or developmental stages. [4] It is known that mutations in the BRCA1 gene are linked to breast cancer. Scientists used conditional gene knockout to delete the BRCA1 allele in mammary gland tissue in mice and found that it plays an important role in tumour suppression. [3]

A specific gene in mouse brain thought to be involved in the onset of Alzheimer's disease which codes for the enzyme cyclin-dependent kinase 5 (Cdk5) was knocked out. Such mice were found to be 'smarter' than normal mice and were able to handle complex tasks more intelligently compared to 'normal' mice bred in the laboratory. [5]

Knockout Mouse Project (KOMP)

Conditional gene knockouts in mice are often used to study human diseases because many genes produce similar phenotypes in both species. For the past 100 years laboratory mouse genetics have been used for this because mice are mammals that are physiologically similar enough to humans to generate qualitative testing. These two have such similar genes that out of 4000 studied genes, only 10 were found in one species but not the other.  All mammals shared the same common ancestor approximately 80 million years ago; technically speaking, all genomes of mammals are comparatively similar. However, in comparison between mice and humans, their protein-coding regions of the genomes are 85% identical and have similarities between 99% of their homologs. These similarities result in similar phenotypes to be expressed between the two species.[8][12] Their genes are very alike to those of humans with 99% having homologs being similar. Along with producing similar phenotypes as well making them very promising candidates for conditional gene knockouts.[8] The goal of KOMP is to create knockout mutations in the embryonic stem cells for each of the 20,000 protein coding genes in mice. [2] The genes are knocked out because this is the best way to study their function and learn more about their role in human diseases. There are two main strategies to conditional gene knockout and those are gene targeting or homologous recombination and gene trapping. Both methods usually have a modified viral vector or a linear fragment as the mode of transportation of the artificial DNA into the target ES cell. The cells then grow in a petri dish for several days and are inserted into the early-stage embryos. Lastly, the embryos are placed into the adult female's uterus where it can grow into its offspring.[9] Some alleles in this project cannot be knocked out using traditional methods and require the specificity of the conditional gene knockout technique. Other combinatorial methods are needed to knockout the last remaining alleles. Conditional gene knockout is a time-consuming procedure and there are additional projects focusing on knocking out the remaining mouse genes. [6] The KOMP project contributor, Oliver Smithies, arguably provided the biggest scientific impact on this gene targeting. Oliver received the Nobel prize for medicine due to a technique allowing the ability to identify functions in genes and how to use the 'knockout' method to delete certain genes. Unfortunately, the pioneer in gene targeting died at the age of 91 on January 10, 2017.[11] The KOMP projected was started in 2006 and is still ongoing today. [7] The KOMP Repository provides incentives to those partaking in the projects to return feedback to them and those who meet specific criteria can be refunded 50% of the cost of their research cells.[10]

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.

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.

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.

Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.

<span class="mw-page-title-main">Cre recombinase</span> Genetic recombination enzyme

Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites. This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be "floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction will be excised as a circular loop of DNA whilst intervening DNA between two loxP sites that are opposingly orientated will be inverted. The enzyme requires no additional cofactors or accessory proteins for its function.

<span class="mw-page-title-main">FLP-FRT recombination</span>

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 µ plasmid of baker's yeast Saccharomyces cerevisiae.

<span class="mw-page-title-main">Gene targeting</span>

Gene targeting is a genetic technique that uses homologous recombination to modify an endogenous gene. The method can be used to delete a gene, remove exons, add a gene and modify individual base pairs. The process of gene targeting provides a way to alter specific genes in order to better identify their biological roles. Gene targeting can be permanent or conditional. Conditions can be a specific time during development / life of the organism or limitation to a specific tissue, for example. Gene targeting requires the creation of a specific vector for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.

RMCE is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes (SSRs). For RMCE, this is achieved by the clean exchange of a preexisting gene cassette for an analogous cassette carrying the "gene of interest" (GOI).

<span class="mw-page-title-main">DUSP5</span> Protein-coding gene in the species Homo sapiens

Dual specificity protein phosphatase 5 is an enzyme that in humans is encoded by the DUSP5 gene.

In molecular cloning and biology, a gene knock-in 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. 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. 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.

<span class="mw-page-title-main">Kelch-like protein 18</span> Protein-coding gene in the species Homo sapiens

Kelch-like protein 18 is a protein that in humans is encoded by the KLHL18 gene.

<span class="mw-page-title-main">KLF17</span> Protein-coding gene in the species Homo sapiens

Krueppel-like factor 17 is a protein that in humans is encoded by the KLF17 gene.

<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">Floxing</span> Sandwiching of a DNA sequence between two lox P sites

In genetics, floxing refers to the sandwiching of a DNA sequence between two lox P sites. The terms are constructed upon the phrase "flanking/flanked by LoxP". Recombination between LoxP sites is catalysed by Cre recombinase. Floxing a gene allows it to be deleted, translocated or inverted in a process called Cre-Lox recombination. The floxing of genes is essential in the development of scientific model systems as it allows researchers to have spatial and temporal alteration of gene expression. Moreover, animals such as mice can be used as models to study human disease. Therefore, Cre-lox system can be used in mice to manipulate gene expression in order to study human diseases and drug development. For example, using the Cre-lox system, researchers can study oncogenes and tumor suppressor genes and their role in development and progression of cancer in mice models.

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">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

<span class="mw-page-title-main">International Mouse Phenotyping Consortium</span>

The International Mouse Phenotyping Consortium (IMPC) is an international scientific endeavour to create and characterize the phenotype of 20,000 knockout mouse strains. Launched in September 2011, the consortium consists of over 15 research institutes across four continents with funding provided by the NIH, European national governments and the partner institutions.

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.

Susan M. Dymecki is an American geneticist and neuroscientist and director of the Biological and Biomedical Sciences PhD Program at Harvard University. Dymecki is also a professor in the Department of Genetics and the principal investigator of the Dymecki Lab at Harvard. Her lab characterizes the development and function of unique populations of serotonergic neurons in the mouse brain. To enable this functional dissection, Dymecki has pioneered several transgenic tools for probing neural circuit development and function. Dymecki also competed internationally as an ice dancer, placing 7th in the 1980 U.S. Figure Skating Championships.

References

  1. 1 2 Varshney, Guarav; Burgess, Shawn (26 October 2013). "Mutagenesis and phenotyping resources in zebrafish for studying development and human disease". Briefings in Functional Genomics. 13 (2): 82–94. doi:10.1093/bfgp/elt042. PMC   3954039 . PMID   24162064.
  2. 1 2 3 Skarnes, William; Rosen, Barry; et al. (2011). "A conditional knockout resource for the genome-wide study of mouse gene function". Nature. 474 (7351): 337–342. doi:10.1038/nature10163. PMC   3572410 . PMID   21677750.
  3. 1 2 3 4 Clarke, Alan (21 March 2000). "Manipulating the germline: its impact on the study of carcinogenesis". Carcinogenesis. 21 (3): 435–441. doi: 10.1093/carcin/21.3.435 . PMID   10688863.
  4. 1 2 3 Zhang, Jian; Zhao, Jing (July 2012). "Conditional gene manipulation: Cre-ating a new biological era". J Zhejiang Univ Sci B. 13 (7): 511–524. doi:10.1631/jzus.b1200042. PMC   3390709 . PMID   22761243.
  5. ↑ "Increased intelligence through genetic engineering". 2007-05-29. Retrieved 2015-05-30.
  6. ↑ Guan, Chunmei; Ye, Chao; Yang, Xiaomei; Gao, Jiangang (2010). "A Review of Current Large-Scale Mouse Knockout Efforts". Genesis. 48 (2): 73–85. doi:10.1002/dvg.20594. PMID   20095055. S2CID   34470273.
  7. ↑ Gondo, Y (2008). "Trends in large-scale mouse mutagenesis: from genetics to functional genomics". Nat. Rev. Genet. 9 (10): 803–810. doi:10.1038/nrg2431. PMID   18781157. S2CID   1435836 . Retrieved 5 November 2015.

8. Austin, C. P., Battey, J. F., Bradley, A., Bucan, M., Capecchi, M., Collins, F. S., Dove, W. F., Duyk, G., Dymecki, S., Eppig, J. T., Grieder, F. B., Heintz, N., Hicks, G., Insel, T. R., Joyner, A., Koller, B. H., Lloyd, K. C., Magnuson, T., Moore, M. W., Nagy, A., ... Zambrowicz, B. (2004). The knockout mouse project. Nature genetics, 36(9), 921–924. https://doi.org/10.1038/ng0904-921

9. Knockout Mice Fact Sheet. (n.d.). Retrieved from https://www.genome.gov/about-genomics/fact-sheets/Knockout-Mice-Fact-Sheet

10. Lloyd K. C. (2011). A knockout mouse resource for the biomedical research community. Annals of the New York Academy of Sciences, 1245, 24–26. https://doi.org/10.1111/j.1749-6632.2011.06311.x

11. Nobel Prize winner Dr. Oliver Smithies to deliver Earl H. Morris Endowed Lecture on July 10. (n.d.). Retrieved from https://medicine.wright.edu/about/article/2009/smithieslecture

12. NIH. (n.d.). Why Mouse Matters. Retrieved from https://www.genome.gov/10001345/importance-of-mouse-genome

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