Knockout mice are produced by disrupting genes in mice through the insertion of artificial DNA, allowing researchers to observe the effects of gene deletion and gain insight into gene function. This document discusses how knockout mice are made via embryonic stem cell manipulation and gene targeting or trapping. It provides the example of cystic fibrosis knockout mice modeling the human disease. While mice are a valuable model for human genetics, there are also limitations such as differences in phenotypes between mice and humans.
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Knockout mice
1. KNOCKOUT MICE: A
MODEL FOR HUMAN
GENETIC DISEASE
AHMAD SHARIFUDDIN, CHUNG CHOON KHAII, SAM KHOO, ZABU KO
2. INTRODUCTION
Certain gene activities are disabled or ‘knocked out’ through replacement or disruption with
artificial pieces of DNA.
The changes in phenotype (appearance, behaviour and other physical and biochemical
characteristics) are observed.
Provides insight into the functions of genes already sequenced.
First produced in 1989 by Mario R. Capecchi, Oliver Smithies and Martin Evans(Nobel Laureates
2007)
Integral in the production of genetic and physical maps.
Production of test subjects for experimental treatments.
3. WHY MICE?
Mammalian vs yeasts(S. cerevisiae), worms(C. elegans) and flies (Drosophila spp.)
Study of complex physiological systems and human diseases
Low cost for maintenance
Short life cycle and many offspring (r-selection)
4. MECHANISM
(Gene Targeting)
Embryonic stem cells are extracted
from early-stage mouse
embryos(Blastocyst).
ES cells are added with targeting
vectors containing a positive and
negative selection cassettes and
homologous regions flanking the
target gene site. (+Neomycin; -Tk
gene)
Targeting vector is transfected into
the target gene through homologous
recombination.
Recombinant transfected cells are
selected by subculture on agar
containing Neomycin and Ganciclovir.
5. MECHANISM
(Gene Trapping)
Random integration of promoter-less
targeting vector.
Non-specific, random binding to
genes expressed in the ES cell.
PCR-based strategies to ascertain
knockout location.
High-throughput method for the
generation of random insertional
mutation.
Generation of gene libraries current
containing mutations in ~60% of all
mice genes.
7. MECHANISM
Generation of chimeric offspring
containing both altered ES cells and
unaltered mouse embryo.
ES cells are injected into mouse
embryo and implanted into surrogate
mother.
Mouse pups produced non-complete
knockout mice.
Chimeric mice are crossbred to
produce full homozygous knockout
mice.
8. CYSTIC FIBROSIS AND THE CTFR
KNOCKOUT MOUSE
Gene targeted knockout of the cystic fibrosis transmembrane conductance regulator (CFTR)
gene in mice.
Loss of expression in CTFR leads to defective cAMP- activatied chloride channel protein.
Generation of common mutations causing CF in humans (p508 deletions).
Provides insight in CF pathology outside clinical and cell culture studies.
Drawbacks in variations of phenotypic expression between mice and humans ranging from
absence to severity(lung infections, gallstone formations, pancreatitis and sweat glands).
11. ADVANTAGES
Provides insight into mutations further than morphological (physiological and behavioural).
Extensively characterized genome sequence strains.
Relatively good models for human disease due to similarities.
Can be used as models for drug and gene therapies.
85% genetic similarity to human genome.
12. DISADVANTAGES AND LIMITATIONS
Developmental lethality limits studies to embryonic development.
Phenotypic differences from mutations between mice and humans.
Ethical concerns.
Difficult to characterize more complex behavioural disorders.
Unable to characterize late stage neurodegenerative disorders.
Lack of diversity of mouse strains cannot capture the diversity of human population.
Cost ineffective.
13. CONCLUSION
Mouse knockout models are invaluable in research into
genotypic-phenotypic relations as well as pathological
understanding of diseases.
The model provides a means of observing gene functions in
living animals.
Mice modelling technology has its advantages and drawbacks
compared to other technologies.
14. REFERENCES
Aguzzi, A., Brandner, S., Sure, U., Rüedi, D. and Isenmann, S. (2008). Transgenic and Knock-out Mice: Models of Neurological Disease. Brain Pathology, 4(1), pp.3-20.
Aguzzi, A., Brandner, S., Sure, U., Rüedi, D. and Isenmann, S. (2008). Transgenic and Knock-out Mice: Models of Neurological Disease. Brain Pathology, 4(1), pp.3-20.
Gonzalez, F. and Kimura, S. (2003). Study of P450 function using gene knockout and transgenic mice.Archives of Biochemistry and Biophysics, 409(1), pp.153-158.
Jidonline.org. (2016). [online] Available at: http://www.jidonline.org/ [Accessed 20 Oct. 2016].
National Human Genome Research Institute (NHGRI). (2016). Knockout Mice Fact Sheet. [online] Available at: https://www.genome.gov/12514551/knockout-mice-fact-sheet/
[Accessed 20 Oct. 2016].
Nature.com. (2016). Figure - Nature Medicine. [online] Available at: http://www.nature.com/nm/journal/v10/n5/fig_tab/nm0504-452_F1.html [Accessed 20 Oct. 2016].
Nobelprize.org. (2016). The Nobel Prize in Physiology or Medicine 2007. [online] Available at:
http://www.nobelprize.org/nobel_prizes/medicine/laureates/2007/illpres/page_three.html [Accessed 20 Oct. 2016].
Skarnes, W. (2005). Two ways to trap a gene in mice. Proceedings of the National Academy of Sciences, 102(37), pp.13001-13002.
Wilke, M., Buijs-Offerman, R., Aarbiou, J., Colledge, W., Sheppard, D., Touqui, L., Bot, A., Jorna, H., De Jonge, H. and Scholte, B. (2011). Mouse models of cystic fibrosis:
Phenotypic analysis and research applications. Journal of Cystic Fibrosis, 10, pp.S152-S171.
Wilke, M., Buijs-Offerman, R., Aarbiou, J., Colledge, W., Sheppard, D., Touqui, L., Bot, A., Jorna, H., De Jonge, H. and Scholte, B. (2011). Mouse models of cystic fibrosis:
Phenotypic analysis and research applications. Journal of Cystic Fibrosis, 10, pp.S152-S171.