KNOCKOUT MICE

KNOCK OUT MICE

• a mouse in which a gene has been deleted/mutated (gene is inactivated)

• specific gene is targeted

• The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.

Researchers who developed the technology for the creation of knockout mice won Nobel Prize in the year 2007

• The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

Knockout vs. Transgenic Mouse?

• Knockout: -gene inactivated -homologous recombination

• Transgenic:- gene(s) added

- can be:- a foreign gene - extra copies of endogenous

gene- mutated endogenous gene

Why Make a Knockout Mouse?

• Determine the function of a particular gene/gene product.• eliminate a specific protein• ask: what can’t that animal do?

What Kinds of Questions ?

• Examine a protein’s role in a biological process- protein X always increased during process Y

• Determine function of a newly identified gene- genome sequencing projects (>30,000 genes)- many of unknown function- some info gained from comparisons

• Protein Families - families of similar yet distinct proteins have the same biochemical function

- all apparently do the same thing- probably have different physiological roles

• Models for Human Diseases - confirm mutation is responsible for disease - studies not possible on humans- experimental therapies

How to make a Knockout?• Have ~ $40,000

• Alter gene of interest in Embryonic Stem (ES) Cells

• Use ES cells to make a mouse

GENERATION OF KNOCKOUT MICE BY HOMOLOGOUS RECOMBINATION• Creating a knockout construct• Introduce the knockout construct into mouse embryonic stem

cells (ES) in culture• Screen ES cells and select those whose DNA includes the new

genes• Implant selected cells into normal mouse embryos , making

“chimeras”• Implant chimeric embryos in pseudopregnant females• Females give birth to chimeric offsprings, which are

subsequently bred to verify transmission of the new gene, producing a mutant mouse line

Knockout construct:

• 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 or that produces an observable change (e.g. colour or fluorescence).

Embryonic Stem (ES) Cells

• isolated from a pre-implantation embryos- from inner cell mass of blastula stage embryo

• cells are undifferentiated• cells are pluripotent

- able to differentiate into many different cell types in embryo

- most importantly germ cells • grown in culture

- need cells to divide but not differentiate- longer time in culture = more differentation

The new DNA sequence is introduced into the embryonic stem cells by electroporation. The natural process of homologous recombination occurs which means 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.

Gene Targeting Technology

• Cells take up exogenous DNA - frequency is very low - can be induced to higher frequency

- chemical, electrical, injection

• In Dividing Cells:- some DNA incorporated into genome

1. Random integration-rare event

2. Homologous Recombination- even more rare

Random Integration:

• DNA incorporated anywhere in genome (not targeted)

• copy number can be very high

• transgenic animals are usually random integrations

Homologous Recombination:

• driven by the DNA sequences (targeted) • rare (1000X less frequent than random)• increased efficiency• results in:

- endogenous DNA replaced with exogenous - specific - targeted- copy number = 1

Targeting Construct for Positive/Negative Selection

• To make targeting construct:- a positive selectable marker flanked by two “arms” of homologous sequence- a negative selectable marker outside one homologous arm

Positive selection markers

• To enrich recombination events• Expression cassettes encoding antibiotic resistance genes

Negative Selection Markers

• Used to enrich for homologous recombination events over random insertions.

• Use of Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene coupled with gancyclovir treatment

Homologous recombinant

Selection Strategy:

• Positive Selection – G418- Neomycin Resistance gene

- confers resistance to G418

• Negative Selection - Gancyclovir- Herpes Simplex Virus Thymidine Kinase (HSV-TK) - sensitive to gancyclovir- selects against random integrants

Making knockout mice

Mutant alleles are introduced by homologous recombination into Embryonic Stem cells.

ES cells containing the knockout mutation are introduced into early mouse embryos.

The resultant mice will be “chimeras” containing tissues derived from both the transplanted ES cells and host cells.

These cells can contribute to both germ cell and somatic cell population

Chimeric mice are mated to assess whether the mutation is incorporated into the germ line

Chimeric mice each heterozygous for the knockout mutation are mated to produce homozygous knockout mice

Procedure for making mixed genotype blastocyst

Breeding scheme for producing knockout mice.

Drawbacks of knockout mice

• About 15% of gene knockouts are developmentally lethal and therefore cannot grow into adult mice. Thus it becomes difficult to determine the gene function in adults.

• Many genes that participate in interesting gene pathways are essential for either mouse development, viability or fertility. Therefore , a traditional knock out of the gene can never lead to the establishment of knockout mouse strain for analysis

Conditional knockout approach

• Delete the gene of interest in a time and space- dependant manner using site-specific recombinases.

• Using these recombinases it is possible to knockout the expression of a gene in a specific mouse tissue or at a specific stage of development or in response to an inducer.

The Cre-loxP System

• 1987- Brian Sauer’s introduction of the Cre-loxP system for temporal control of transgenic gene expression

• 1995- K Rajewsky demonstrated “inducible gene targetting in

mice” using the Cre-loxP conditional knockout

• Cre recombinase is a site specific integrase isolated from P1 bacteriophage

• Catalyses recombiniation between two of its consensus DNA recognition sites known as the loxP sites which are 34 bp in length

• Two 13 bp palindromic sequences that flank a central sequence of 8 bp which determines the directionality of the loxP site

Spatial and Temporal knockout is achieved by the choice of promoter used to drive the Cre-gene expression:

• Several mouse lines each expressing Cre from a promoter that is either tissue-specific, cell specific, developmentally specific,or responsive to an exogenous agent like tetracycline are now available.

• Thus, several promoter specific mouse models can be generated

Knockout vs. Knock in

• In contrast to knockout in which a gene or part of gene is deleted, knockin is the replacement of the gene by mutant version of the same gene using homologous recombination.

• Knockin is very useful while establishing a disease model of a specific disease related mutation in human gene.

Mice - Models of Human DiseasesAlthough the human is the mammal we are generally most

interested in learning more about, it is also the one animal we cannot use for genetic experiments for obvious ethical reasons

Mice naturally develop conditions that mimic human disease, such as cardiovascular disease, cancer and diabetes

Mouse are a favorite model for human disease because it has a relatively low cost of maintenance and a generation time that measures only nine weeks

Developments in molecular biology and stem cell biology have allowed researchers to create custom-made mice through gene targeting in mouse embryonic stem (ES) cells

Certain diseases that afflict only humans, such as cystic fibrosis and Alzheimer's can also be induced by manipulating the mouse genome and environment

Knockout Mice to study genetic diseases

• Knockout mice make good model systems for investigating the nature of genetic diseases and the efficacy of different types of treatment and for developing effective gene therapies to cure these often devastating diseases

• For instance, the knockout mice for CFTR gene show symptoms similar to those of humans with cystic fibrosis

Knockout mice and cancer

• Rb gene was knocked out but instead of eye tumours the animals suffered from pituitary gland and thyroid gland tumours

• It was later found that a second gene protected the eye cells from cancer and both mutations are required for tumours to form

Research using knockout mice• Examples of research in which knockout mice have been

useful include studying and modelling different kinds of-obesity-heart disease-diabetes-arthritis-Parkinson’s disease

• Knockout mice also offer a biological and scientific context in which drugs and other therapies can be developed and tested.

http://www.dnalc.org/view/16856-Animation-41-DNA-is-only-the-beginning-for-understanding-the-human-genome-.html