KNOCKOUT MICE: A MODEL FOR HUMAN GENETIC DISEASEAHMAD SHARIFUDDIN, CHUNG CHOON KHAII, SAM KHOO, ZABU KO

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.

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)

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.

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.

Gene Targeting vs Gene Trapping

MECHANISMGeneration 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.

CYSTIC FIBROSIS AND THE CTFR KNOCKOUT MOUSEGene 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).

Cystic Fibrosis in Mice

List of Different Knockout Mutations in Mice

ADVANTAGESProvides 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.

DISADVANTAGES AND LIMITATIONSDevelopmental 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.

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.

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