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Knockout mice

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Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Animal studies, cftr knockout mouse, embryo, embryonic stem cells, foreign DNA, gene targeting, gene therapy, gene trapping, genetic engineering, genotype, homologous recombination, homozygous knockouts, inactivated gene, knock out, knockout model, p53 gene, phenotype, reporter gene, targeted recombination, wild type.

Background
  • A knockout mouse is a genetically engineered mouse used for research. Knockout mice have specific genes that are "knocked out" or deactivated.
  • Genes are made of specific DNA sequences and provide the instructions for making proteins and other molecules. Thus, genes control the development and function of an organism. Genes are passed down as cells divide and from parents to their offspring.
  • To create a knockout mouse, scientists either replace or deactivate an existing gene with a gene targeting construct. Targeting constructs may contain "foreign" DNA from other organisms and are designed to contain sequences that are similar, or homologous, to the mouse DNA. There are several types of targeting constructs. Some insert a piece of foreign DNA into the gene, which interrupts the gene sequence. If designed properly, this interruption may inactivate the gene. Other targeting constructs are designed to replace a gene (or critical gene sequence) with foreign DNA. Some targeting constructs are designed so that an entire gene (or a critical part of the gene) is removed completely. Depending on the type of procedure used, the constructs may target either specific genes or random DNA sequences.
  • An organism's genetic makeup (genotype) influences its behavior and physical characteristics (phenotype). When a gene is knocked out, this changes the organism's genotype. As a result, the phenotype of a knockout mouse may differ from a natural, or "wild type" mouse.
  • This type of research allows scientists to learn what specific genes do. When a gene is deactivated, the function of that gene may be revealed by the characteristics of the knockout mouse.
  • Mice and humans share about 85% of their genes. Thus, many discoveries made in mice are often applicable to humans. In addition, much of the research done in mice would be unethical or impossible in humans. Therefore, using mice can help researchers learn more about the safety of certain procedures before trying them in humans.
  • Genetically engineered mice may also provide insight into some diseases and inherited disorders. Using knockout mice, scientists have learned more about many different diseases, including cystic fibrosis, obesity, heart disease, arthritis, blindness, Parkinson's disease, muscular dystrophy, aging, and cancer. Understanding the mechanisms behind these diseases is important because it may help researchers develop new ways to treat and prevent certain illnesses.

Methods
  • Harvesting stem cells: To make a knockout mouse, scientists change the mouse's genetic information in the very early stages of development. Scientists take embryonic stem cells from a mouse embryo that is just a few days old. Researchers use embryonic stem cells because they are unspecialized cells that are able to grow and develop into any cell type. When a gene is knocked out in an embryonic stem cell, the effects can potentially be seen in any tissue in an adult mouse. Embryonic stem cells can be grown in the laboratory and stored in frozen stocks for future use.
  • Inserting artificial genes: To produce knockout mice, scientists must insert artificial DNA into the embryonic stem cells. Two different methods can be used: gene targeting or gene trapping. Both of these procedures are done in cells that are grown in a laboratory.
  • Gene targeting: During gene targeting, which is also called homologous recombination, scientists pinpoint the specific gene they want to knock out inside the nucleus of an embryonic stem cell. The nucleus is a small organ-like structure that contains the cell's genetic makeup.
  • Researchers are able to manipulate this specific gene by using a gene-targeting construct. This construct contains some DNA that is identical to the mouse's existing gene. This construct also contains DNA that inactivates the gene by replacing or interrupting the targeted gene.
  • When the targeting construct is inserted, the identical DNA sequences may "change places" or "recombine" with the targeted gene. The mouse DNA is exchanged for the DNA in the targeting construct. If designed properly, the targeting construct will inactivate the desired gene.
  • Scientists use this method to produce knockout mice because they can efficiently "turn off" specific genes. However, this method is only efficient if scientists already know the DNA sequence of the gene they want to turn off.
  • Gene trapping: Unlike gene targeting, gene trapping does not involve choosing and locating a specific gene to knockout. Instead, gene trapping involves randomly turning off a single gene.
  • During the procedure, a special type of artificial DNA is inserted into the embryonic stem cell. This reporter gene prevents the cell's RNA from splicing properly. As a result, it randomly prevents one gene from functioning properly, effectively knocking out that gene.
  • One advantage of gene trapping is that scientists do not need to know the DNA sequences of specific genes in order to knock them out.
  • However, gene trapping is less efficient and specific than gene targeting. This is because not every successful insertion of artificial DNA causes a gene to lose its function. Also, some genes may never get knocked out using this random process, and it is time-consuming for researchers to determine if a gene has actually been turned off. Other genes do not have active functions in the cells, which mean researchers will not know if they have been knocked out.
  • Stem cells are inserted into an embryo: Once the artificial DNA is inserted (using one of the two methods listed above), the embryonic stem cells are grown in a laboratory for several days. Then the cells are injected into mouse embryos. Next, the embryos are inserted into a female mouse's uterus. Finally, the female gives birth to the knockout pups.
  • Resulting pups(baby mice): Once the pups are born, researchers evaluate the mice's physical and biochemical characteristics to determine the knocked-out gene's function. The researchers compare the knockout mice to unaltered mice. By identifying characteristics that differ between the knockout mice and unaltered mice, researchers can learn about the function of the gene that was turned off.

Research
  • Gene functions: Knockout mice are studied to provide information about the function of specific genes. Using knockout mice, researchers have been able to identify and understand the functions of many important genes that are also present in humans. Individual genes can change or influence an organism's appearance, behavior, and/or biochemical makeup. For example, researchers have identified genes that influence aggression, alcoholism, drug addiction, and maternal behavior.
  • Medical conditions: Learning about specific gene functions is important because it provides insight into different types of inherited disorders and medical conditions. For instance, knockout mice have helped scientists learn about genes that are associated with cystic fibrosis, obesity, heart disease, arthritis, blindness, Parkinson's disease, muscular dystrophy, aging, and cancer.
  • Knockout studies have helped scientists identify a cancer gene, called the p53 gene. This gene helps prevent tumors, including cancerous tumors, from growing in the body. This gene has been shown to help prevent cells from growing and dividing uncontrollably. Individuals who are born with inactive p53 genes have a condition called Li-Fraumeni syndrome, which leads to an increased risk of breast cancer, bone cancers, and blood cancers. Knockout studies have allowed scientists to learn more about how this disease works.
  • A cystic fibrosis knockout mouse model has helped researchers learn more about a fatal genetic disease called cystic fibrosis. With the use of knockout mice, scientists have discovered that cystic fibrosis is caused by a defect in the gene that produces a protein called cystic fibrosis transmembrane conductance regulator (CFTR). Cystic fibrosis knockout mice have helped scientists develop new approaches to treat, and possibly prevent, cystic fibrosis.
  • Knockout mice have also helped researchers identify the gene that is associated with glaucoma, a group of eye disorders that increase the pressure inside the eyeball and may lead to blindness.

Implications
  • Knockout models may help researchers understand the functions of specific genes, which is important because it can help researchers understand what causes inherited disorders and diseases. As a result, many knockout studies have helped scientists develop and test new drug therapies and treatments for these conditions in humans.
  • For example, researchers are currently studying a type of gene therapy that involves inserting a normal p53 gene into a patient that is missing the gene. Researchers hope that by replacing this specific gene, it may help treat or prevent cancer because the p53 gene has been shown to help the body fight tumors.

Limitations
  • An estimated 15% of gene knockout studies cause the mouse to die before it can develop into an adult mouse. When a mouse dies before adulthood, it makes it difficult to understand a gene's function in relation to human health. This is because some genes function differently during development than they do during adulthood.
  • Sometimes, an inactivated gene in a mouse produces no observable change in the animal's phenotype. In other cases, the observed change may be different in mice than it is in humans. For instance, an abnormal or absent p53 gene increases the risk of certain types of cancers. However, mice and humans with abnormal or absent p53 genes develop cancer in different parts of the body.

Safety




Future research
  • Studies with knockout mice are ongoing. Scientists aim to identify and understand the functions of many important mouse genes that are also present in humans. These studies also help researchers gain a better understanding of different types of inherited disorders and medical conditions.

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography
  1. Canale-Zambrano JC, Poffenberger MC, Cory SM. Intestinal phenotype of variable-weight cystic fibrosis knockout mice. Am J Physiol Gastrointest Liver Physiol. 2007 Jul;293(1):G222-9.
  2. Conarello SL, Jiang G, Mu J, et al. Glucagon receptor knockout mice are resistant to diet-induced obesity and streptozotocin-mediated beta cell loss and hyperglycaemia. Diabetologia. 2007 Jan;50(1):142-50. Epub 2006 Nov 28.
  3. Garcia MC< Wernstedt I, Berndtsson A, et al. Mature-onset obesity in interleukin-1 receptor I knockout mice. Diabetes. 2006 May;55(5):1205-13.
  4. Houghtaling S, Granvile L, Akkari Y, et al. Heterozygosity for p53 (Trp53+/-) accelerates epithelial tumor formation in fanconi anemia complementation group D2 (Fancd2) knockout mice. Cancer Res. 2005 Jan 1;65(1):85-91.
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  8. Salleh MN, Ismail P, Abdullah AS, et al. Gene expression profiling of p53(+/-) knockout and wild-type mice following diethylstilbestrol administration. IUBMB Life. 2004 Jul;56(7):409-16.
  9. Stephenson J.Knockout science: chubby mice provide new insights into obesity. JAMA. 1999 Oct 27;282(16):1507-8.

Copyright © 2011 Natural Standard (www.naturalstandard.com)


The information in this monograph is intended for informational purposes only, and is meant to help users better understand health concerns. Information is based on review of scientific research data, historical practice patterns, and clinical experience. This information should not be interpreted as specific medical advice. Users should consult with a qualified healthcare provider for specific questions regarding therapies, diagnosis and/or health conditions, prior to making therapeutic decisions.

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