Knockout mouse

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Knockout_mouse 

A knockout mouse, or knock-out mouse, is a genetically modified mouse (Mus musculus) 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.

Mice are currently the laboratory animal species most closely related to humans 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 2007 Nobel Prize in Physiology or Medicine. Aspects of the technology for generating knockout mice, and the mice themselves have been patented in many countries by private companies.

Use

A laboratory mouse in which a gene affecting hair growth has been knocked out (left) is shown next to a normal lab mouse.

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, 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.

Strains

A knockout mouse (left) that is a model for obesity, compared with a normal mouse

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 and/or inducing apoptosis. Humans born with mutations that deactivate the p53 gene have 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 according to their physical characteristics or behaviours.

Procedure

The procedure for making mixed-genotype blastocyst

Breeding scheme for producing knockout mice. Blastocysts containing cells, that are both wildtype and knockout cells, are injected into the uterus of a foster mother. This produces offspring that are either wildtype and coloured the same colour as the blastocyst donor (grey) or chimera (mixed) and partially knocked out. The chimera mice are crossed with a normal wildtype mouse (grey). This produces offspring that are either white and heterozygous for the knocked out gene or grey and wildtype. White heterozygous mice can subsequently be crossed to produce mice that are homozygous for the knocked out gene.

There are several variations to the procedure of producing knockout mice; the following is a typical example.

1. The gene to be knocked out is isolated from a mouse gene library. 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 the gene inoperable. Usually, the new sequence is also given a marker gene, a gene that normal mice don't have and that confers resistance to a certain toxic agent (e.g., neomycin) or that produces an observable change (e.g. colour or fluorescence). In addition, a second gene, such as herpes tk+, is also included in the construct in order to accomplish a complete selection.
2. Embryonic stem cells are isolated from a mouse blastocyst (a very young embryo) and grown in vitro. For this example, we will take stem cells from a white mouse.
3. The new sequence from step 1 is introduced into the stem cells from step 2 by electroporation. By the natural process of homologous recombination some of the electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene. The chances of a successful recombination event are relatively low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes – they are said to be heterozygous. Cells that were transformed with a vector containing the neomycin resistance gene and the herpes tk+ gene are grown in a solution containing neomycin and Ganciclovir in order to select for the transformations that occurred via homologous recombination. Any insertion of DNA that occurred via random insertion will die because they test positive for both the neomycin resistance gene and the herpes tk+ gene, whose gene product reacts with Ganciclovir to produce a deadly toxin. Moreover, cells that do not integrate any of the genetic material test negative for both genes and therefore die as a result of poisoning with neomycin.
4. The embryonic stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene from step 1. For example, the unaltered cells can be killed using a toxic agent to which the altered cells are resistant.
5. The knocked-out embryonic stem cells from step 4 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse. The blastocysts now contain two types of stem cells: the original ones (from the grey mouse), and the knocked-out cells (from the white mouse). These blastocysts are then implanted into the uterus of female mice, where they develop. The newborn mice will therefore be chimeras: some parts of their bodies result from the original stem cells, other parts from the knocked-out stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst.
6. Some of the newborn chimera mice will have gonads derived from knocked-out stem cells, and will therefore produce eggs or sperm containing the knocked-out gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the knocked-out gene in all their cells. These mice do not retain any grey mouse DNA and are not chimeras, however they are still heterozygous.
7. When these heterozygous offspring are interbred, some of their offspring will inherit the knocked-out gene from both parents; they carry no functional copy of the original unaltered gene (i.e. they are homozygous 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 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 mutations. The lack of adult mice limits studies to embryonic development and 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.

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 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.

Asymmetry

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Asymmetry

Asymmetry is the absence of, or a violation of, symmetry (the property of an object being invariant to a transformation, such as reflection). Symmetry is an important property of both physical and abstract systems and it may be displayed in precise terms or in more aesthetic terms. The absence of or violation of symmetry that are either expected or desired can have important consequences for a system.

In organisms

Due to how cells divide in organisms, asymmetry in organisms is fairly usual in at least one dimension, with biological symmetry also being common in at least one dimension.

Louis Pasteur proposed that biological molecules are asymmetric because the cosmic [i.e. physical] forces that preside over their formation are themselves asymmetric. While at his time, and even now, the symmetry of physical processes are highlighted, it is known that there are fundamental physical asymmetries, starting with time.

Asymmetry in biology

Main articles: Symmetry in biology and Left-right asymmetry (biology)

Asymmetry is an important and widespread trait, having evolved numerous times in many organisms and at many levels of organisation (ranging from individual cells, through organs, to entire body-shapes). Benefits of asymmetry sometimes have to do with improved spatial arrangements, such as the left human lung being smaller, and having one fewer lobes than the right lung to make room for the asymmetrical heart. In other examples, division of function between the right and left half may have been beneficial and has driven the asymmetry to become stronger. Such an explanation is usually given for mammal hand or paw preference (handedness), an asymmetry in skill development in mammals. Training the neural pathways in a skill with one hand (or paw) may take less effort than doing the same with both hands.

Nature also provides several examples of handedness in traits that are usually symmetric. The following are examples of animals with obvious left-right asymmetries:

Male fiddler crab, Uca pugnax

• Most snails, because of torsion during development, show remarkable asymmetry in the shell and in the internal organs.
• Male fiddler crabs have one big claw and one small claw.
• The narwhal's tusk is a left incisor which can grow up to 10 feet in length and forms a left-handed helix.
• Flatfish have evolved to swim with one side upward, and as a result have both eyes on one side of their heads.
• Several species of owls exhibit asymmetries in the size and positioning of their ears, which is thought to help locate prey.
• Many animals (ranging from insects to mammals) have asymmetric male genitalia. The evolutionary cause behind this is, in most cases, still a mystery.

As an indicator of unfitness

• Certain disturbances during the development of the organism, resulting in birth defects.
• Injuries after cell division that cannot be biologically repaired, such as a lost limb from an accident.

Since birth defects and injuries are likely to indicate poor health of the organism, defects resulting in asymmetry often put an animal at a disadvantage when it comes to finding a mate. For example, a greater degree of facial symmetry is seen as more attractive in humans, especially in the context of mate selection. In general, there is a correlation between symmetry and fitness-related traits such as growth rate, fecundity and survivability for many species. This means that, through sexual selection, individuals with greater symmetry (and therefore fitness) tend to be preferred as mates, as they are more likely to produce healthy offspring.

In structures

Pre-modern architectural styles tended to place an emphasis on symmetry, except where extreme site conditions or historical developments lead away from this classical ideal. To the contrary, modernist and postmodern architects became much more free to use asymmetry as a design element.

While most bridges employ a symmetrical form due to intrinsic simplicities of design, analysis and fabrication and economical use of materials, a number of modern bridges have deliberately departed from this, either in response to site-specific considerations or to create a dramatic design statement.

Some asymmetrical structures

• 
  Eastern span replacement of the San Francisco – Oakland Bay Bridge
  
  
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  Puente de la Mujer
  
  
• 
  Auditorio de Tenerife
  
  
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  Blohm & Voss BV 141 aircraft
  
  
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  A proa, a form of outrigger canoe

In fire protection

In fire-resistance rated wall assemblies, used in passive fire protection, including, but not limited to, high-voltage transformer fire barriers, asymmetry is a crucial aspect of design. When designing a facility, it is not always certain, that in the event of fire, which side a fire may come from. Therefore, many building codes and fire test standards outline, that a symmetrical assembly, need only be tested from one side, because both sides are the same. However, as soon as an assembly is asymmetrical, both sides must be tested and the test report is required to state the results for each side. In practical use, the lowest result achieved is the one that turns up in certification listings. Neither the test sponsor, nor the laboratory can go by an opinion or deduction as to which side was in more peril as a result of contemplated testing and then test only one side. Both must be tested in order to be compliant with test standards and building codes.

In mathematics

See also: Symmetry in mathematics

In mathematics, asymmetry can arise in various ways. Examples include asymmetric relations, asymmetry of shapes in geometry, asymmetric graphs et cetera.

Lines of symmetry

When determining whether an object is asymmetrical, look for lines of symmetry. For instance, a square has four lines of symmetry, while a circle has infinite. If a shape has no lines of symmetry, then it is asymmetrical, but if an object has any lines of symmetry, it is symmetrical.

Asymmetric Relation

An asymmetric relation is a binary relation ${\displaystyle R}$ defined on a set of elements such that if ${\displaystyle aRb}$ holds for elements ${\displaystyle a}$ and ${\displaystyle b}$, then ${\displaystyle bRa}$ must be false. Stated differently, an asymmetric relation is characterized by a necessary absence of symmetry of the relation in the opposite direction.

Inequalities exemplify asymmetric relations. Consider elements ${\displaystyle a}$ and ${\displaystyle b}$. If ${\displaystyle a}$ is less than ${\displaystyle b}$ (${\displaystyle a<b}$), then ${\displaystyle a}$ cannot be greater than ${\displaystyle b}$ (${\displaystyle a\ngtr b}$). This highlights how the relations "less than", and similarly "greater than", are not symmetric.

In contrast, if ${\displaystyle a}$ is equal to ${\displaystyle b}$ (${\displaystyle a=b}$), then ${\displaystyle b}$ is also equal to ${\displaystyle a}$ (${\displaystyle b=a}$). Thus the binary relation "equal to" is a symmetric one.

Asymmetric Tensors

In general an Asymmetric tensor is defined by the change of signs${\displaystyle (-/+)}$ of its solution under the interchange of two indexes.

The Epsilon-tensor is an example of an asymmetric tensor. It is defined as: ${\displaystyle {ϵ}_{ijk}=\{\begin{array}{ll}1& \text{if }(i,j,k)\in \{(123),(231),(312)\}\\ -1& \text{if }(i,j,k)\in \{(213),(321),(132)\}\\ 0& \text{else}\end{array}}$

,with ${\displaystyle i,j,k\in \{1,2,3\}}$. For even or uneven permutations of the indexes the tensor is either 1 or -1.

In chemistry

Certain molecules are chiral; that is, they cannot be superposed upon their mirror image. Chemically identical molecules with different chirality are called enantiomers; this difference in orientation can lead to different properties in the way they react with biological systems.

In physics

Asymmetry arises in physics in a number of different realms.

Thermodynamics

The original non-statistical formulation of thermodynamics was asymmetrical in time: it claimed that the entropy in a closed system can only increase with time. This was derived from the Second Law (either of the two, Clausius' or Lord Kelvin's statement, can be used since they are equivalent) and using the Clausius' Theorem (see Kerson Huang ISBN 978-0471815181). The later theory of statistical mechanics, however, is symmetric in time. Although it states that a system significantly below maximum entropy is very likely to evolve towards higher entropy, it also states that such a system is very likely to have evolved from higher entropy.

Particle physics

Symmetry is one of the most powerful tools in particle physics, because it has become evident that practically all laws of nature originate in symmetries. Violations of symmetry therefore present theoretical and experimental puzzles that lead to a deeper understanding of nature. Asymmetries in experimental measurements also provide powerful handles that are often relatively free from background or systematic uncertainties.

Parity violation

Main article: parity (physics)

Until the 1950s, it was believed that fundamental physics was left-right symmetric; i.e., that interactions were invariant under parity. Although parity is conserved in electromagnetism, strong interactions and gravity, it turns out to be violated in weak interactions. The Standard Model incorporates parity violation by expressing the weak interaction as a chiral gauge interaction. Only the left-handed components of particles and right-handed components of antiparticles participate in weak interactions in the Standard Model. A consequence of parity violation in particle physics is that neutrinos have only been observed as left-handed particles (and antineutrinos as right-handed particles).

In 1956–1957 Chien-Shiung Wu, E. Ambler, R. W. Hayward, D. D. Hoppes, and R. P. Hudson found a clear violation of parity conservation in the beta decay of cobalt-60. Simultaneously, R. L. Garwin, Leon Lederman, and R. Weinrich modified an existing cyclotron experiment and immediately verified parity violation.

CP violation

Main article: CP-violation

After the discovery of the violation of parity in 1956–57, it was believed that the combined symmetry of parity (P) and simultaneous charge conjugation (C), called CP, was preserved. For example, CP transforms a left-handed neutrino into a right-handed antineutrino. In 1964, however, James Cronin and Val Fitch provided clear evidence that CP symmetry was also violated in an experiment with neutral kaons.

CP violation is one of the necessary conditions for the generation of a baryon asymmetry in the early universe.

Combining the CP symmetry with simultaneous time reversal (T) produces a combined symmetry called CPT symmetry. CPT symmetry must be preserved in any Lorentz invariant local quantum field theory with a Hermitian Hamiltonian. As of 2006, no violations of CPT symmetry have been observed.

Baryon asymmetry of the universe

Main article: baryogenesis

The baryons (i.e., the protons and neutrons and the atoms that they comprise) observed so far in the universe are overwhelmingly matter as opposed to anti-matter. This asymmetry is called the baryon asymmetry of the universe.

Isospin violation

Isospin is the symmetry transformation of the weak interactions. The concept was first introduced by Werner Heisenberg in nuclear physics based on the observations that the masses of the neutron and the proton are almost identical and that the strength of the strong interaction between any pair of nucleons is the same, independent of whether they are protons or neutrons. This symmetry arises at a more fundamental level as a symmetry between up-type and down-type quarks. Isospin symmetry in the strong interactions can be considered as a subset of a larger flavor symmetry group, in which the strong interactions are invariant under interchange of different types of quarks. Including the strange quark in this scheme gives rise to the Eightfold Way scheme for classifying mesons and baryons.

Isospin is violated by the fact that the masses of the up and down quarks are different, as well as by their different electric charges. Because this violation is only a small effect in most processes that involve the strong interactions, isospin symmetry remains a useful calculational tool, and its violation introduces corrections to the isospin-symmetric results.

In collider experiments

Because the weak interactions violate parity, collider processes that can involve the weak interactions typically exhibit asymmetries in the distributions of the final-state particles. These asymmetries are typically sensitive to the difference in the interaction between particles and antiparticles, or between left-handed and right-handed particles. They can thus be used as a sensitive measurement of differences in interaction strength and/or to distinguish a small asymmetric signal from a large but symmetric background.

• A forward-backward asymmetry is defined as A_FB=(N_F-N_B)/(N_F+N_B), where N_F is the number of events in which some particular final-state particle is moving "forward" with respect to some chosen direction (e.g., a final-state electron moving in the same direction as the initial-state electron beam in electron-positron collisions), while N_B is the number of events with the final-state particle moving "backward". Forward-backward asymmetries were used by the LEP experiments to measure the difference in the interaction strength of the Z boson between left-handed and right-handed fermions, which provides a precision measurement of the weak mixing angle.
• A left-right asymmetry is defined as A_LR=(N_L-N_R)/(N_L+N_R), where N_L is the number of events in which some initial- or final-state particle is left-polarized, while N_R is the corresponding number of right-polarized events. Left-right asymmetries in Z boson production and decay were measured at the Stanford Linear Collider using the event rates obtained with left-polarized versus right-polarized initial electron beams. Left-right asymmetries can also be defined as asymmetries in the polarization of final-state particles whose polarizations can be measured; e.g., tau leptons.
• A charge asymmetry or particle-antiparticle asymmetry is defined in a similar way. This type of asymmetry has been used to constrain the parton distribution functions of protons at the Tevatron from events in which a produced W boson decays to a charged lepton. The asymmetry between positively and negatively charged leptons as a function of the direction of the W boson relative to the proton beam provides information on the relative distributions of up and down quarks in the proton. Particle-antiparticle asymmetries are also used to extract measurements of CP violation from B meson and anti-B meson production at the BaBar and Belle experiments.