Knockout mouse
Encyclopedia
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.
Knockout mice are important animal model
s 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.
Mice are currently the most closely related laboratory animal species
to human
s for which the knockout technique can easily be applied. They are widely used in knockout experiments, especially those investigating genetic questions that relate to human physiology
. Gene knockout
in rats
is much harder and has only been possible since 2003.
The first recorded knockout mouse was created by Mario R. Capecchi
, Martin Evans
and Oliver Smithies
in 1989, for which they were awarded the Nobel Prize
for Medicine in 2007. Aspects of the technology for generating knockout mice, and the mice themselves have been patent
ed in many countries by private companies.
Examples of research in which knockout mice have been useful include studying and modeling different kinds of cancer
, obesity
, heart disease
, diabetes
, arthritis
, substance abuse
, anxiety
, aging
and Parkinson's disease
. Knockout mice also offer a biological and scientific context in which drugs and other therapies can be developed and tested.
Millions of knockout mice are used in experiments each year.
There are several thousand different strains of knockout mice.
Many mouse models are named after the gene that has been inactivated. For example, the p53
knockout mouse is named after the p53 gene
which codes for a protein that normally suppresses the growth of tumours by arresting cell division
. Humans born with mutations that deactivate the p53 gene suffer from Li-Fraumeni syndrome
, a condition that dramatically increases the risk of developing bone cancers, breast cancer
and blood cancers at an early age. Other mouse models are named, often with creative flair, according to their physical characteristics or behaviours.
There are several variations to the procedure of producing knockout mice; the following is a typical example.
A detailed explanation of how knockout (KO) mice are created is located at the website of the Nobel Prize in Physiology or Medicine
2007.
There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived. Generally cells derived from strain 129 are used. This specific strain is not suitable for many experiments (e.g., behavioural), so it is very common to backcross
the offspring to other strains. Some genomic loci have been proven very difficult to knock out. Reasons might be the presence of repetitive sequences, extensive DNA methylation
, or heterochromatin
. The confounding presence of neighbouring 129 genes on the knockout segment of genetic material has been dubbed the "flanking-gene effect". Methods and guidelines to deal with this problem have been proposed.
Another limitation is that conventional (i.e. non-conditional) knockout mice develop in the absence of the gene being investigated. At times, loss of activity during development may mask the role of the gene in the adult state, especially if the gene is involved in numerous processes spanning development. Conditional/inducible mutation approaches are then required that first allow the mouse to develop and mature normally prior to ablation of the gene of interest.
Another serious limitation is a lack of evolutive adaptations in knockout model that might occur in wild type animals after they naturally mutate. For instance, erythrocyte-specific coexpression of GLUT1
with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C
.
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
mouse
House mouse
The house mouse is a small rodent, a mouse, one of the most numerous species of the genus Mus.As a wild animal the house mouse mainly lives associated with humans, causing damage to crops and stored food....
in which researchers have inactivated, or "knocked out," an existing gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
by replacing it or disrupting it with an artificial piece of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. The loss of gene activity often causes changes in a mouse's phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
, which includes appearance, behavior and other observable physical and biochemical characteristics.
Knockout mice are important animal model
Animal model
An animal model is a living, non-human animal used during the research and investigation of human disease, for the purpose of better understanding the disease without the added risk of causing harm to an actual human being during the process...
s for studying the role of genes which have been sequenced
Sequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
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.
Mice are currently the most closely related laboratory animal species
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
to human
Human
Humans are the only living species in the Homo genus...
s for which the knockout technique can easily be applied. They are widely used in knockout experiments, especially those investigating genetic questions that relate to human physiology
Human physiology
Human physiology is the science of the mechanical, physical, bioelectrical, and biochemical functions of humans in good health, their organs, and the cells of which they are composed. Physiology focuses principally at the level of organs and systems...
. Gene knockout
Gene knockout
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
in rats
Brown Rat
The brown rat, common rat, sewer rat, Hanover rat, Norway rat, Brown Norway rat, Norwegian rat, or wharf rat is one of the best known and most common rats....
is much harder and has only been possible since 2003.
The first recorded knockout mouse was created by Mario R. Capecchi
Mario Capecchi
Mario Renato Capecchi is an Italian-born American molecular geneticist and a co-winner of the 2007 Nobel Prize in Physiology or Medicine for discovering a method for introducing homologous recombination in mice employing embryonic stem cells, with Martin Evans and Oliver Smithies...
, Martin Evans
Martin Evans
Sir Martin John Evans FRS is a British scientist who, with Matthew Kaufman, was the first to culture mice embryonic stem cells and cultivate them in a laboratory in 1981...
and Oliver Smithies
Oliver Smithies
Oliver Smithies is a British-born American geneticist and Nobel laureate, credited with the invention of gel electrophoresis in 1955, and the simultaneous discovery, with Mario Capecchi and Martin Evans, of the technique of homologous recombination of transgenic DNA with genomic DNA, a much more...
in 1989, for which they were awarded the Nobel Prize
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...
for Medicine in 2007. Aspects of the technology for generating knockout mice, and the mice themselves have been patent
Patent
A patent is a form of intellectual property. It consists of a set of exclusive rights granted by a sovereign state to an inventor or their assignee for a limited period of time in exchange for the public disclosure of an invention....
ed in many countries by private companies.
Use
Knocking out the activity of a gene provides information about what that gene normally does. Humans share many genes with mice. Consequently, observing the characteristics of knockout mice gives researchers information that can be used to better understand how a similar gene may cause or contribute to disease in humans.Examples of research in which knockout mice have been useful include studying and modeling different kinds of cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
, obesity
Obesity
Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems...
, heart disease
Heart disease
Heart disease, cardiac disease or cardiopathy is an umbrella term for a variety of diseases affecting the heart. , it is the leading cause of death in the United States, England, Canada and Wales, accounting for 25.4% of the total deaths in the United States.-Types:-Coronary heart disease:Coronary...
, diabetes
Diabetes mellitus
Diabetes mellitus, often simply referred to as diabetes, is a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced...
, arthritis
Arthritis
Arthritis is a form of joint disorder that involves inflammation of one or more joints....
, substance abuse
Substance abuse
A substance-related disorder is an umbrella term used to describe several different conditions associated with several different substances .A substance related disorder is a condition in which an individual uses or abuses a...
, anxiety
Anxiety
Anxiety is a psychological and physiological state characterized by somatic, emotional, cognitive, and behavioral components. The root meaning of the word anxiety is 'to vex or trouble'; in either presence or absence of psychological stress, anxiety can create feelings of fear, worry, uneasiness,...
, aging
Ageing
Ageing or aging is the accumulation of changes in a person over time. Ageing in humans refers to a multidimensional process of physical, psychological, and social change. Some dimensions of ageing grow and expand over time, while others decline...
and Parkinson's disease
Parkinson's disease
Parkinson's disease is a degenerative disorder of the central nervous system...
. Knockout mice also offer a biological and scientific context in which drugs and other therapies can be developed and tested.
Millions of knockout mice are used in experiments each year.
Strains
Many mouse models are named after the gene that has been inactivated. For example, the p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
knockout mouse is named after the p53 gene
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
which codes for a protein that normally suppresses the growth of tumours by arresting cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
. Humans born with mutations that deactivate the p53 gene suffer from Li-Fraumeni syndrome
Li-Fraumeni syndrome
Li-Fraumeni syndrome is an extremely rare autosomal dominant hereditary disorder. It is named after Frederick Pei Li and Joseph F. Fraumeni, Jr., the American physicians who first recognized and described the syndrome. Li-Fraumeni syndrome greatly increases susceptibility to cancer...
, a condition that dramatically increases the risk of developing bone cancers, breast cancer
Breast cancer
Breast cancer is cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. Cancers originating from ducts are known as ductal carcinomas; those originating from lobules are known as lobular carcinomas...
and blood cancers at an early age. Other mouse models are named, often with creative flair, according to their physical characteristics or behaviours.
Procedure
- The gene to be knocked out is isolated from a mouse gene libraryLibrary (biology)In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning...
. Then a new DNA sequence is engineered which is very similar to the original gene and its immediate neighbour sequence, except that it is changed sufficiently to make it inoperable. Usually, the new sequence is also given a marker geneMarker geneA marker gene is a gene used in molecular biology to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. There are two types of marker genes: a selectable marker and a marker for screening.-Selectable marker:...
, a gene that normal mice don't have and that confers resistance to a certain toxic agent or that produces an observable change (e.g. colour or fluorescence). The chances of a successful recombination event are relatively low, so the majority of altered cells will have the gene changed for only one of the two relevant chromosomes - they are said to be heterozygousZygosityZygosity refers to the similarity of alleles for a trait in an organism. If both alleles are the same, the organism is homozygous for the trait. If both alleles are different, the organism is heterozygous for that trait...
. - From a mouse blastocystBlastocystThe blastocyst is a structure formed in the early embryogenesis of mammals, after the formation of the morula. It is a specifically mammalian example of a blastula. It possesses an inner cell mass , or embryoblast, which subsequently forms the embryo, and an outer layer of cells, or trophoblast,...
(a very young embryoEmbryoAn embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
consisting of a ball of undifferentiated cells with surrounding extra-embryonic cells), stem cellStem cellThis article is about the cell type. For the medical therapy, see Stem Cell TreatmentsStem cells are biological cells found in all multicellular organisms, that can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells...
s are isolated; these can be grown in vitroIn vitroIn vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
. For this example, we will take a stem cell from a white mouse. - The stem cells from step 2 are combined with the new sequence from step 1. This is done via electroporationElectroporationElectroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
(using electricity to transfer the DNA across the cell membrane). Some of the electroporated stem cells will incorporate the new sequence into their chromosomeChromosomeA chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s in place of the old gene; this is called homologous recombinationHomologous recombinationHomologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
. The reason for this process is that the new and the old sequences are very similar. Using the marker gene from step 1, those stem cells that actually did incorporate the new sequence can be quickly isolated from those that did not. - The stem cells from step 3 are inserted into a mouse blastocystBlastocystThe blastocyst is a structure formed in the early embryogenesis of mammals, after the formation of the morula. It is a specifically mammalian example of a blastula. It possesses an inner cell mass , or embryoblast, which subsequently forms the embryo, and an outer layer of cells, or trophoblast,...
. For this example, we use blastocysts from a grey mouse. These blastocysts are then implanted into the uterusUterusThe uterus or womb is a major female hormone-responsive reproductive sex organ of most mammals including humans. One end, the cervix, opens into the vagina, while the other is connected to one or both fallopian tubes, depending on the species...
of female mice, to complete the pregnancy. The blastocysts contain two types of stem cells: the original ones (grey mouse), and the newly engineered ones (white mouse). The newborn mice will therefore be chimeras: parts of their bodies result from the original stem cells, other parts result from the engineered stem cells. Their furs will show patches of white and grey, with white patches derived from the stem cells and grey patches from the recipient blastocyst. - Newborn mice are only useful if the newly engineered sequence was incorporated into the germ cellGerm cellA germ cell is any biological cell that gives rise to the gametes of an organism that reproduces sexually. In many animals, the germ cells originate near the gut of an embryo and migrate to the developing gonads. There, they undergo cell division of two types, mitosis and meiosis, followed by...
s (egg or sperm cellsSpermatozoonA spermatozoon is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote...
). These new mice are crossed with others of the wild type for offspring that are all white. These mice still contain one functional copy of the gene and must be further inbred to produce mice that carry no functional copy of the original gene (i.e. are homozygousZygosityZygosity refers to the similarity of alleles for a trait in an organism. If both alleles are the same, the organism is homozygous for the trait. If both alleles are different, the organism is heterozygous for that trait...
for that allele).
A detailed explanation of how knockout (KO) mice are created is located at the website of the Nobel Prize in Physiology or Medicine
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine administered by the Nobel Foundation, is awarded once a year for outstanding discoveries in the field of life science and medicine. It is one of five Nobel Prizes established in 1895 by Swedish chemist Alfred Nobel, the inventor of dynamite, in his will...
2007.
Limitations
The National Institutes of Health discusses some important limitations of this technique.
While knockout mouse technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. This problem is often overcome through the use of conditional mutationsMutationIn molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
. The lack of adult mice limits studies to embryonic developmentEmbryogenesisEmbryogenesis is the process by which the embryo is formed and develops, until it develops into a fetus.Embryogenesis starts with the fertilization of the ovum by sperm. The fertilized ovum is referred to as a zygote...
and often makes it more difficult to determine a gene's function in relation to human healthHealthHealth is the level of functional or metabolic efficiency of a living being. In humans, it is the general condition of a person's mind, body and spirit, usually meaning to be free from illness, injury or pain...
. In some instances, the gene may serve a different function in adults than in developing embryos.
Knocking out a gene also may fail to produce an observable change in a mouse or may even produce different characteristics from those observed in humans in which the same gene is inactivated. For example, mutations in the p53 gene are associated with more than half of human cancers and often lead to tumours in a particular set of tissues. However, when the p53 gene is knocked out in mice, the animals develop tumours in a different array of tissues.
There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived. Generally cells derived from strain 129 are used. This specific strain is not suitable for many experiments (e.g., behavioural), so it is very common to backcross
Backcrossing
Backcrossing is a crossing of a hybrid with one of its parents or an individual genetically similar to its parent, in order to achieve offspring with a genetic identity which is closer to that of the parent...
the offspring to other strains. Some genomic loci have been proven very difficult to knock out. Reasons might be the presence of repetitive sequences, extensive DNA methylation
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
, or heterochromatin
Heterochromatin
Heterochromatin is a tightly packed form of DNA, which comes in different varieties. These varieties lie on a continuum between the two extremes of constitutive and facultative heterochromatin...
. The confounding presence of neighbouring 129 genes on the knockout segment of genetic material has been dubbed the "flanking-gene effect". Methods and guidelines to deal with this problem have been proposed.
Another limitation is that conventional (i.e. non-conditional) knockout mice develop in the absence of the gene being investigated. At times, loss of activity during development may mask the role of the gene in the adult state, especially if the gene is involved in numerous processes spanning development. Conditional/inducible mutation approaches are then required that first allow the mouse to develop and mature normally prior to ablation of the gene of interest.
Another serious limitation is a lack of evolutive adaptations in knockout model that might occur in wild type animals after they naturally mutate. For instance, erythrocyte-specific coexpression of GLUT1
GLUT1
Glucose transporter 1 , also known as solute carrier family 2, facilitated glucose transporter member 1 is a protein that in humans is encoded by the SLC2A1 gene...
with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C
Vitamin C
Vitamin C or L-ascorbic acid or L-ascorbate is an essential nutrient for humans and certain other animal species. In living organisms ascorbate acts as an antioxidant by protecting the body against oxidative stress...
.
See also
- Genetically modified organismGenetically modified organismA genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
- GeneticsGeneticsGenetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
- HumouseHumouseA humouse is an immunodeficient mouse reconstituted with a human immune system. Although conventional mouse models have allowed for an increased understanding of mammalian immune systems, sometimes this knowledge cannot be directly applied to humans due to biological differences between the two...
- International Knockout Mouse ConsortiumInternational Knockout Mouse ConsortiumThe International Knockout Mouse Consortium is a scientific endeavour to produce a collection of mouse embryonic stem cell lines that together lack every gene in the genome, and then to distribute the cells to scientific researchers to create knockout mice to study...
- Knockout moss
- OncomouseOncomouseThe OncoMouse or Harvard mouse is a type of laboratory mouse that has been genetically modified using modifications designed by Philip Leder and Timothy A Stewart of Harvard University to carry a specific gene called an activated oncogene. The activated oncogene significantly increases the mouse’s...
External links
- Texas A&M Institute for Genomic Medicine (TIGM) - The web site for ordering ES cells and mice generated by TIGM
- Studying Gene Function: Creating Knockout Mice - a review from the Science Creative Quarterly
- The Knock Out Mouse Project (KOMP) Data Coordination website - The public interface for information on the status of the genes included in the KOMP initiative.
- The Knock Out Mouse Project (KOMP) Repository website- The web site for ordering ES cells, vectors, and mice generated by the KOMP project
- Mouse Genome Informatics (MGI) website - community model organism database for the laboratory mouse
- Homologous Recombination Method (and Knockout Mouse)