Cell Biology for Knockout Mice

Cell Biology for Knockout Mice Experiments With Diabetes

Genetic engineering holds some real promise for curing the diseases that afflict mankind and for extending human lives. To further these genetic investigations, scientists use knockout mice in an effort to determine what a gene normally does by observing the effects of its functional elimination. These knockout mice experiments have already provided scientists with some new insights into the etiologies of many diseases and these genetic engineering techniques hold true promise for the future. This paper provides a review of the relevant peer-reviewed and scholarly literature to determine the mechanisms and techniques of the knockout mice experiments, including how they are performed and how they affect the mice at the cellular level in terms of cell structure and/or function. A discussion of some of the experiments in which knockout mice have been used in the study of diabetes is followed by a summary of the research in the conclusion.

Review and Discussion

Background and Overview.

One of the unfortunate consequences of living in the 21st century is the fact that people today stand a better chance of contracting diabetes than ever before. In fact, over the past few decades, the incidence of diabetes has approached epidemic levels and there are currently 177 million persons with diabetes in the world (World Health Organization 2005). The basis for this increased incidence of diabetes has been directly correlated with many aspects of modern lifestyles, particularly a paucity of exercise combine wit an unhealthy diet characterized by fast food and vending machine snacks (Alonso-Magdalena et al. 106). According to the World Health Organization:

Diabetes causes about 5% of all deaths globally each year;

80% of people with diabetes live in low and middle income countries;

Most people with diabetes in low and middle income countries are middle-aged (45-64), not elderly (65-plus); and,

Diabetes deaths are likely to increase by more than 50% in the next 10 years without urgent action.

There has been a dramatically increased incidence for other pathologies after World War II as well, including cancer, reproductive impairment, and neurodegenerative diseases; all of these conditions have been attributed to the increase of endocrine-disrupting chemicals in the environment (Barondes).

Other environmental factors are also being studied to determine if there is a relationship between environment factors and the incidence of diabetes in the 21st century. In this regard, Lindberg and her colleagues (2007) report that, "Arsenic is a worldwide water contaminant, and chronic exposure has been associated with a large number of health effects, such as different forms of cancer, skin lesions, vascular diseases, liver -- and neurotoxicity, and diabetes mellitus. Studies with GSTO1 knockout mice showed that they still reduced arsenic (V) species, but to a lesser extent (~ 20% of that found in wild-type mice)" (1081). Likewise, according to Stevens and his colleagues (2007), "One of the defining characteristics of life in the modern world is the altered patterns of light and dark in the built environment made possible by use of electric power" (1357). The growing of evidence has determined the underlying mechanisms responsible for photo-transduction in the retina that provide environmental control of circadian and other neurobehavioral responses and the composition and functioning of the clock physiology that exert genetic control of the endogenous rhythms (Stevens et al. 1357). These authors adds that, "There is limited but thus far generally consistent evidence in support of the hypothesis that altered lighting can play a role in breast cancer causation, and there is growing interest in a lighting and/or sleep connection to other conditions such as prostate cancer, obesity, diabetes and depression" (Stevens et al. 1358). While the light detection for the regulatory function performed by the circadian, neuroendocrine, and neurobehavioral systems appear to be mediated principally by intrinsically photoreceptive retinal ganglion cells, experiments using melanopsin knockout mice have demonstrated that the classic rod and cone visual photoreceptors nevertheless appear to have some role in modulating these responses, findings that may ultimately help researchers develop more effective clinical interventions, vaccines and cures (Stevens et al. 1358).

Taken together, the foregoing suggests that environmental factors are responsible for the near-epidemic levels of diabetes around the world and scientists are using such genetically engineered knockout mice to help further their research into the etiology of diabetes and other diseases through a process known as gene targeting, in which an existing gene variant (variant 1) is replaced by an alternative variant (variant 2) of the same gene (Barondes, 2003). Gene targeting is accomplished by taking the variant 1 and "cutting it out" its normal position on a chromosome and substituting variant 2 in the same precise location; what results from this genetically engineering is a line of mice with variant 2 instead of variant 1 (Barondes). In the majority of cases, variant 2 is a man-made gene variant that has been prepared in a test tube by chemical manipulation of isolated copies of variant 1, with the chemical manipulation being intended to effect a specific change in the function of variant 1 by substituting or removing some of its nucleotides (Barondes).

In some cases, variant 2 is designed with a major flaw that causes it be nonfunctional, and variant 2 is then inserted into the DNA of embryos by a technique that exchanges it for variant 1; the descendants of embryos whose variant 1 was replaced by the nonfunctional variant 2 are known as "knockout" mice because the function of the gene has been "knocked out" (Barondes). As Leshner (1999) advises, "In the past few years, we have been able to create genetically altered knockout mice, which lack one or more of these receptors. Studies of the drug-responsiveness and behavior of these mice have illuminated the complexity and the interconnectedness of brain mechanisms. For example, experiments with these knockout mice have demonstrated that the pleasurable effects of cocaine remain despite the absence of the dopamine transporter, a molecule previously thought to be the primary mediator of these effects" (22).

In 2001, Mario Capecchi, Martin Evans, and Oliver Smithies received the Albert Lasker Basic Medical Research Award for their work that led to the development of knockout mice (Lauerman 2002). This author adds that, "Knockout mice, developed in 1989, are now commonly used by medical researchers to replace mice genes with faulty human genes and thereby give human diseases to mice. The development of knockout mice has provided researchers with a powerful tool for testing drugs and with insight into how diseases develop. Although Capecchi, Evans, and Smithies did not actually develop the first knockout mice, their research made such an achievement possible" (Lauerman 312).

While the use of knockout mice is commonplace today, their development was an extremely complicated process that required the better part of the 1980s because of the state of technology of the day: "Snipping out a mouse gene was simple enough by the early 1980s, but replacing that gene with another one was considered an impossible task. Whenever DNA was introduced into a mammalian cell, the new DNA would insert itself at a random site rather than in the place where the gene had been snipped out" (Lauerman 313). At the time, a number of researchers believed that these challenges were insurmountable; however, Capecchi and Smithies independently developed a process called homologous recombination, in which new DNA lines up with a specific site on a chromosome and either replaces the existing gene or is added as a second copy (Lauerman 313). According to this author, "In 1982, Capecchi proved that mammalian cells already had a mechanism for incorporating introduced DNA into specific locations on existing chromosomes, which raised the possibility that scientists could do the same" (Lauerman 313).

The possibility of doing the same and actually doing it were, of course, dramatically different issues and Capecchi and Smithies found it difficult to determine which techniques worked because successes were so rare; nevertheless, these researchers, again working separately, developed methods whereby it was possible to determine if the cells had reached their appropriate destination on the DNA strand. Smithies is credited with first discovering that a cell that had been engineered successfully, and his publication of the discovery in 1985 established the groundwork for future experiments that targeted DNA segments into a mammalian chromosome (Lauerman 313). While this was an innovation, the method developed by Smithies was exceedingly time consuming and Capecchi is credited with developing more efficient techniques to identify cells that had had their DNA altered correctly, research that eventually resulted in methods of incorporating the DNA that were more likely to be successful (Lauerman 313).

In 1988, Capecchi developed a general technique to facilitate the growth of cells that have properly incorporated introduced DNA and to eliminate those that have not incorporated the desired DNA; during the period when Capecchi and Smithies were working separately on the genetic component of the problem, Evans was working on how best to use the mice needed for such experiments (Lauerman 313). According to this author, "In the early 1980s, Evans isolated embryonic stem cells in mice. Stem cells are cells that can develop into other forms of cells; Evans's cells could develop into entire mice. Evans eventually began altering the genetic material in the stem cells, creating mice that had genetic material from other creatures and could pass that material on to their offspring" (313). These findings, together with the research conducted separately by Capecchi and Smithies, enabled several teams of researchers to develop knockout mice (Lauerman 313).

In 2007, Evans received the Nobel Prize for medicine for these discoveries and the development of knockout mice that could be used to help scientists better understand and possibly cure diseases such as cystic fibrosis, heart disease, diabetes and cancer (Briton wins Nobel Prize 4). According to the editors of Environmental Health Perspectives (2005), the Comparative Mouse Genomics Centers Consortium (CMGCC) already has 54 transgenic or knockout mouse models developed at varying stages of construction and characterization (with regard to genotyping and phenoytping) for use as single nucleotide polymorphisms mice (Mouse models to improve understanding of the biological significance of human polymorphisms, 2005). Today, knockout mice are providing an increasing amount of valuable scientific information concerning the potential roles of genes in biological and behavioral processes and in the pathophysiology of many diseases which are discussed further below.

Mechanisms and Techniques of Knockout Mice Experiments.

Knockout mice are generally used to find out what a gene normally does; the mice are using for this purpose by observing the effects of functional elimination of certain genes on its functioning; however, just as the development of the knockout mice themselves was enormously challenging, this process is also not as straightforward as it sounds because the gene may be involved in a number of interacting biological processes (Barondes). As Bowers (2000) also points out, the fundamental requirements for creating such a transgenic or "knockout" mouse include:

Identifying and isolating the candidate gene of interest from its original organism (i.e., from the DNA of the organism's cells); and,

Selecting a suitable promoter that is placed adjacent to the transgene. The choice of promoter by the scientist depends on its location in the brain and the time in which (i.e., prenatal or postnatal) the transgene must be expressed (175).

While scientists are currently able to control the expression of the transgene through the careful selection of the promoter (promoters are stretches of DNA associated with a specific gene that guide the expression of the gene to specific areas in the brain and turn the expression of the gene 'on' either prenatally or postnatally), one limitation of the transgenic technique is its inability to target the integration of the transgene to its natural location on the chromosome (Bowers 175). As this author points out, "The site of integration is unique for each microinjection, and the transgene can be randomly inserted anywhere on any chromosome. This outcome can result in the disruption of a sequence of one of the host animal's own genes (i.e., known as insertional mutagenesis), producing changes in behavior that could mistakenly be attributed to the transgene itself" (Bowers 175).

In addition, scientists are still unable to control the number of integrated copies of the transgene, and the presence of additional copies of a gene does not necessarily indicate an increased overexpression of the gene being studied (Bowers 175). According to Bowers, "To control for this, the existence of more than one founder and consequently more than one line of mice for each transgene is desirable. The site of integration and level of expression will differ in each founder, and transgenic mice that descend from the same founder will share the same chromosomal integration site. If each transgenic line displays the same changes in behavior, it is more likely that it is attributable to the transgene" (175). Furthermore, the human body has also been shown to compensate for the loss of certain genes by increasing or decreasing the activities of related genes (Barones).

Despite these constraints to research, knockout mice have traditionally been used to study developmental processes and as models to study the etiology of human diseases and the environmental factors that may contribute to their incidence (Bowers 175). Some of the information that scientists have derived from their knockout experiments with mice have already yielded some impressive results. For example, a noteworthy discovery was made from the knockout of a gene that encodes a protein that can be divided to produce two different brain peptides (Barondes).

Termed "hypocretins" because they are manufactured in the hypothalamus, these two peptides are also called orexins (from the Greek word for "appetite" -- the same root that is used in "anorexia") in some cases because they are thought to increase appetite (120). In an effort to investigate the behavioral functions of the hypocretins, Chemelli, Sinton, Elmquist et al. (1999) knocked out the hypocretin gene, thereby deleting both of these peptides; when the researchers videotaped the knockout mice to monitor their movements, they determined that the mice kept falling asleep for short periods, an abnormal pattern of behavior is characteristic of narcolepsy (cited in Barondes at 121).

While these observations were being made, a role for hypocretins in narcolepsy was identified elsewhere by Lin, Hungs, and Mignot (2001). This team of researchers had been investigating a genetic form of narcolepsy in a line of Doberman pinschers by using the same gene-hunting techniques that resulted in the identification of gene variants that cause Alzheimer's disease and determined that this variant encodes an inactive form of a receptor for one of the hypocretins (hypocretin receptor-2). Based on this discovery, the Chemelli experiment knocked out this hypocretin receptor in mice and confirmed that its absence -- like the absence of the hypocretins themselves-causes narcolepsy (cited in Barondes at 121).

Studies Using Knockout Mice in the Study of Diabetes.

In recent years, knockout mice have been used to identify epitopes on several autoimmune disease-related antigens such as collagen and cartilage glycoprotein, the autoantigens associated with arthritis, glutamic acid decarboxylase and insulin associated with type 1 diabetes, nicotinic acetylcholine receptor involved in myasthenia gravis, RO60 (SS-a) lupus-related antigen, myelin basic protein, myelin olygodendrocyte glycoprotein and proteolipid protein implicated in the development of multiple sclerosis; in addition, the use of certain knockout mice has been shown to be an effective tool for the identification of T-cell epitopes on malarial parasites and mycobacterial antigens that could be used to generate a new type of more effective subunit vaccines (Chapoval and David 2003:245). More recent studies of specific types of knockout mice immune responses to tumor-associated proteins has provided researchers with new insights that have identified epitopes of potential clinical value for design of anticancer vaccines (Chapoval and David 246).