KNOCKOUT MICE

By-Lovnish ThakurASU2014010100099Integrated Biotech 3rd semCourse-Genetic Engineering; Gmo’s

What are knockout mice?

A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” 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 and thus provides valuable information on the function of the gene.

Nobel Prize in Physiology in 2007

Mario R. Capecchi, Sir Martin J. Evans & Oliver Smithies

"for their discoveries of principles for introducing specific gene modifications in mice by the use of

embryonic stem cells".

Procedure

1. The gene to be knocked out is isolated from a mouse gene library

2. Then a new DNA sequence is engineered which is very similar to the original gene and its immediate neighbor sequence, except that it is changed sufficiently to make it inoperable.

3. From a mouse morula stem cells are isolated; these can be grown in vitro. For this example, we will take a stem cell from a white mouse.

4. The stem cells from step 3 are combined with the new sequence from step 2.

• via electroporation• selected using antibiotic

5. The stem cells from step 4 are inserted into mouse blastocyst cells.

6. Blastocysts are then implanted into the uterus of female mice, to complete the pregnancy.

7. The blastocysts contain two types of stem cells: • The original ones• The newly engineered ones

8. 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.

9. Newborn mice are only useful if the newly engineered sequence was incorporated into the germ cells (egg or sperm cells).

10. Cross these new mice with others and watch for offspring that are all white. These are then further inbred to produce mice that carry no functional copy of the original gene.

Transgenic vs Knockout mice

A transgenic mouse typically expresses one or more copies of a gene (cDNA)that is integrated into its genome in a random fashion.

knockout mouse is a mouse in which both alleles of a gene are deleted in a targeted fashion by homologous recombination

Knockout vs Knockin

knockout in which a gene or part of a gene is

deleted

Importance of knockout mice in biology & medicine

1. provides valuable information about the function(s) of that gene.

2. genes are common between humans and mice, knockout mice gives researchers information about the function of that gene in humans.

3. useful in studying and modeling different kinds of• cancer,• Obesity• heart disease• diabetes• arthritis,• anxiety,• aging and• Parkinson disease. 4. Knockout mice also offer a biological context in which drugs and other therapies

can be developed and tested

SOME EXAMPLES

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

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

Limitations• 15 percent of gene knockouts are developmentally lethal, which

means that the genetically altered embryos cannot grow into adult mice.

• lack of adult mice- 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

• There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived.

References

• http://www.cellmigration.org/resource/komouse/komouse_approaches.shtml

• Cell Migration Consortium Transgenic & Knockout Mouse Initiative

• http://learn.genetics.utah.edu/content/tech/transgenic/

• http://www.eucomm.org/information/protocols/

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