Knockout Mice Technology | Submitted By : Wasman ID : 11136007Course ID : BTE 402 |

Knockout Mice Technology | Submitted By : Wasman ID : 11136007Course ID : BTE 402 |

Modern medicine is the product of centuries of experiences in dealing with various diseases and disorders related to the human physiology. From the discovery of vaccination to the discovery of antibiotics, each medical breakthrough highlighted the success of humans in dealing with the prevailing medical issues of that time. With the discovery of the structure of DNA by Crick and Watson in the early 1950s, the focus on medical research has shifted to the understanding of the genetic cause of a particular disease. Because of this, we are now able to understand the pathophysiology behind much of the diseases that has been a mystery to us before. An example of this is cancer, which was initially untreatable, but now thanks to our ever improving understanding of the genetic and biochemical mechanisms of the disease we are almost a step closer to achieving a reliable cure.
In order to understand the progression of a particular disease accurately, scientists need to recreate the symptoms in a controlled environment (e.g. a laboratory) within a living organism. Such organisms are known as model organisms, because they serve as a live model which allows us to explore the workings of the disease. Due to various ethical issues, using humans as model organisms are out of the question, so scientists have looked towards other organisms to fulfil this purpose. Different organisms have been used for different research purposes, with notable examples being the use of organisms such as yeasts, worms and flies for studying the cell cycle and many developmental processes. For genetic diseases, however, a genetically similar organism to humans are required, since the results will be different in organisms of different genetic makeup. Over the past century, the mouse has developed into the premier mammalian model system for genetic research. Scientists from a wide range of biomedical fields have gravitated to the mouse because of its close genetic (approximately 95%) and physiological similarities to humans, as well as the ease with which its genome can be manipulated and analyzed. Mice, therefore, are a far better tool for probing the immune, endocrine, nervous, cardiovascular, skeletal and other complex physiological systems that mammals share and the impact of these systems in the event of a disease. Adding to the mouse's appeal as a model for biomedical research is the animal's relatively low cost of maintenance and its ability to quickly multiply, reproducing as often as every nine weeks. The resulting genomic manipulation of mice have yielded many different strains of mice with different properties and characteristics. One of these is the knockout mouse.
What are knockout mice?
A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable physical and biochemical characteristics. The resulting phenotypic change in the mouse can be used as an indicator for the gene’s normal role in mouse and, by extrapolation, humans. Developed in 1989 by Mario R. Capecchi, Martin Evans and Oliver Smithies, the trio were awarded a Nobel Prize for Physiology in 2007 for their groundbreaking work which enabled scientist to study a range of genetic disorders (such as Parkinson’s disease, cancer and diabetes) as well as lifestyle problems (such as obesity and substance abuse).
Since a single knockout mouse can be used as a model for one particular disease, several thousand different mice strains have been developed since they were first introduced. At the forefront of this technology is The Jackson Laboratory, a publicly supported national repository for mouse models in Bar Harbor, Maine. It has played a crucial role in the development of the mouse into the leading model for biomedical research and is currently the leading distributor of knockout mice strains with almost 2700 different strains in their database alone. A majority of these are knockout mice strains, which are named according to the gene that has been knocked out. Some of these include: * The Cftr knockout mouse: This particular strain has helped advance research into cystic fibrosis, the most common fatal genetic disease in the United States today, occurring in approximately one of every 3,300 live births. In this particular strain, the Cftr gene has been disabled which thereby eliminates the formation of the CFTR carrier protein in cell membranes; the protein regulates the passage of salts and water in and out of cells. Studies with the Cftr knockout have shown that the disease results from a failure to clear certain bacteria from the lung, which leads to mucus retention and subsequent lung disease. These mice have become models for developing new approaches to correct the CF defect and cure the disease. * The p53 knockout mouse: It has a disabled Trp53 tumor suppressor gene, which restricts the manufacturing of that particular protein which plays and important role in arresting cell division. This makes the mouse highly susceptible to various cancers, including lymphomas and osteosarcomas. The mouse has emerged as an important model for human Li-Fraumeni syndrome, a form of familial breast cancer.
Creating a Knockout Mouse:
Figure 1Vector construction of a knockout mouse
Figure [ 1 ]Vector construction of a knockout mouse
Knockout mice can be created by two main processes, which are gene targeting and gene trapping. Although both have their different strength, the gene targeting method is widely preferred as it is more precise and highly efficient as opposed to the more random method of gene trapping. The process of creating a knockout mouse by the method of gene targeting involves two main processes. The first step involves the construction of the vector carrying the desired characteristic(s) to be expressed whereas the other step involves inserting the vector into embryonic stem cells of a mouse and inducing it to develop. However that is just a brief description of an otherwise complex process.
As mentioned earlier, the first step of the process involves the construction of the vector. The vector consists of two regions of homology at either end, the gene of interest, and two additional genes to allow for quality control in the process. The target gene is isolated from a mouse gene library. The homology regions are located at either end of the vector and are complementary to specific sites on the genome surrounding the gene of interest, whereas the target gene is sequenced using the mouse gene library as reference. The extra genes serve as selection markers, which are classified as positive and negative selection markers based on their mode of action. The positive selection markers allows the cell to survive in toxic conditions if it has been successfully implemented whereas the negative markers kills them in a normally non-toxic environment. Because of this, the positive selection marker is always inserted within the homology regions along with the target gene. The negative selection marker is generally not required, but maybe added for greater quality control.
Figure 2: Overall illustration of a knockout mouse strain production process
Figure [ 2 ]: Overall illustration of a knockout mouse strain production process
Figure.1 and 2 illustrates the method by which the vector is inserted and the modified ES (embryonic stem) cells are selected. The entire process is carried out in vitro, with the ES cells derived from any mouse grown in a media with the vector. The cells take in the vector after it has been subjected to electroporation (Fig. 2).Afterwards, the desired gene in the vector is inserted into the target DNA via homologous recombination (Fig. 1). In this process, the gene of interest is swapped with the DNA in the genome after the vector is aligned with the targeted locus. By mechanisms that are poorly understood but are similar to what occurs during meiosis and mitosis when homologous chromosomes align along the metaphase plane, the engineered construct finds the targeted gene and recombination takes place within the homologous sequences. This may take place anywhere within the flanking DNA sequences and the exact location is determined by the cell itself. To ensure insertion of the gene, the positive selection marker (in this case, neomycin) is included, which provides the recombinant ES cell with resistance to neomycin so that non-recombinant cells are selected out when grown in a neomycin media (fig. 1). For further control, a negative selection marker is used, which in this case is a thymidine kinase (tk) gene. It prevents the insertion of the vector in the genome at the wrong place and ensures recombination by giving the non-recombinant cell thymidine kinase activity. This makes it sensitive to gancyclovir. The end product of this selection is the recombinant cell, otherwise known as the knocked-out cell.
After a pure population of recombinant ES cells are obtained, the cells from the culture are inserted into a blastocyst extracted from a mouse with fur color that is different from that of the ES cell donor (a white mouse in fig. 2). These blastocysts are then implanted into the uterus of female mice, where they develop. The mother mice gives birth to a range of mice, some with normal fur color while others being chimeras: mice with both grey and white fur as some parts of their bodies result from the original stem cells (white) and the other parts from the knocked-out stem cells(grey).
As all cell types are affected, some of the chimeric mice will have gonads that produce sperm or egg cells containing the knocked-out gene. When they are crossbred with a normal white mouse, some of their offspring will have one copy of the knocked-out gene in all their cells. These mice will be entirely grey and are not chimeras, however their cells will be heterozygous from the recombination. These offspring are interbred to produce a strain of mice which will inherit both knocked-out alleles from the parents (i.e. a homozygous knock-out mouse).

Applications of knock-out mice:
The production of mice with specific deletion of targeted genes has provided a useful tool in understanding the mechanisms underlying behavior of various diseases. As mentioned before, the deletion of the genes provides us with the view of the phenotypic effects associated with such a phenomenon. These models are impossible to observe in a culture dish, and therefore these mice are ideal for the task. The results obtained from these mice has been used in the study of diseases caused by the same gene deletion in humans such as Type 1 Diabetes ,cancers and Cystic Fibrosis and Beta Thalassemia to name a few. The hope is that by better understanding how a certain gene contributes to a particular disease, researchers can then take the knowledge a step further and look for drugs that act on that gene.
Another notable application of such mice is in the research of causative factors behind diseases associated with lifestyle changes. Examples of such problems include obesity, heart problems and Type 2 diabetes. These diseases are fairly common nowadays, owing to our current living environment and our way of life. The aim of such studies is to establish a link between the genetic factors and the environment effects in the progression of the disorder. Figure 3 shows an artificially developed obese knockout mouse compared to a regular lab mouse.
Wild-type mice has been used by scientist for the purpose of drug development for many years. With knockout mice, in which specific disease symptoms can be recreated, researchers can discover new blockbuster therapies that act more effectively and can work at the genomic level before the drug is even produced. The concept is simple: since most drugs act as antagonists that block the activity of a particular protein target, a knockout mouse can be used as a genetic model of an ideal drug that is 100% specific and 100% potent at the target of interest, and delivered to 100% of tissues and cells.
Limitations and Drawbacks:
Despite all the benefits offered by knockout mice technology, it has several notable drawbacks. One of the most important of these is the relatively high failure rate of knockout mice embryos in thriving. This is attributed to the fact that most gene knockouts are developmentally lethal, and as such the altered embryo cannot grow into adult mice. Because of this, studies are generally restricted to embryonic development and this often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos.
Although the similarities between human and mouse genome are great, knockout mice may also fail to produce any phenotypic change or in certain cases, may even display totally different characteristics in contrast to the ones observed in humans when the same gene is disrupted. An example of this is the knockout mice strain in which the p53 gene is deactivated. p53 is an important component of the signal transduction system and also a tumor suppressing protein. Because of this, mutations in the p53 gene (which leads to inactivation or overstimulation of the protein manufacturing process) are associated with more than half of human cancers and often lead to tumors in a particular set of tissues. However, when a similar effect was recreated in knockout mice, the result were totally different as the tumors developed in a different set of tissues.
Aside from the aforementioned issues, other drawbacks related to the use of knockout mice models include limitations such as the spontaneous exhibition of the characteristic to be observed in the mice in the absence of the gene being investigated, which masks the role of the gene in the adult state, and the lack of evolutionary adaptations that might occur in wild type animal due to spontaneous mutation (such as the co-expression of certain proteins when an important synthesis pathway has been disabled by a mutation). From an economic viewpoint, producing custom knockout mice is very expensive, with typical cost ranging anywhere from $3,000 to as much as $30,000 per mouse. Coupled with this is the high maintenance cost for these genetically altered animals, as they require special facilities to keep them at optimal conditions for research purposes.
Despite these drawbacks, knockout mice offer one of the most powerful means now available for studying gene function in a living animal. Such studies will accelerate efforts to translate newfound knowledge of the human and mouse genomes into better strategies for diagnosing, treating and preventing human disease.

References: * http://www.scq.ubc.ca/studying-gene-function-creating-knockout-mice/ * http://www.bio.davidson.edu/courses/genomics/method/homolrecomb.html#KO * http://genome.wellcome.ac.uk/doc_WTD021038.html * http://www.cellmigration.org/resource/komouse/komouse_approaches.shtml * http://www.genome.gov/10005834 * http://www.genome.gov/12514551 * http://genetics.thetech.org/original_news/news63 * http://physrev.physiology.org/content/78/4/1131.full * http://www.nature.com/scitable/topicpage/scientists-can-analyze-gene-function-by-deleting-6526138