Pharmacogenomics 3 Flashcards
why are strains of mice useful transgenic models ?
because the strains of mice are homogenous and so if a genetic modification is induced its effects can be determined with greater uniformity
what has transgenic mice had a significant impact on ?
on all aspects of biomedical and biological research
it accounts for the increase in the number of animals used in research
what can be seen in transgenic mice?
overexpression of a foreign gene– add more to the mouse genome
expression of markers under the control of specific promoters - using markers to determine where specific ccells were
- beta galactosidase= detection by enzyme reaction
- green fluorescent protein= detection by fluroescence microscopy
how are transgenic mice produced ?
1- fertile eggs removed from superovulated mouse
2- transgene is directly injected into male pronucleus
3- injected eggs implanted into surrogate female mice
4- offspring screened for presence of transgene in their genome
5- transgenic offspring used to breed transgenic line
what alterations to the DNA can be carried out to produce transgenic mice ?
can take a mutant dominant gene and introduce it into a mouse to determine its affects
can add an extra bit of DNA into mouse- this is the simplest method of producing a transgenic mouse
what is the standard transgenic approach ?
1- transgene DNA is microinjected into the male pronucleus of a fertilised murine oocyte
2- injected oocytes are transferred to a 0.5 day pseudopregnant recipient mouse
3- offspring are screened for the transgene by DNA analysis
what are the issues associated with the standard transgenic approach ?
no control over where the gene will integrate and you dont know how many copies will integrate -
its integration may disrupt the function of another gene
what is the gene-targeted transgenic approach ?
1- isogenic transgene DNA is introduced into embryonic stem cells
2- drug selection is used and the surviving colonies are screened for the transgene
3- characterised targeted cells are microinjected into 3.5 day mouse blastocysts
4- blastocysts are transferred to a 2.5 day pseudopregnant recipient ouse
5- chimeric offspring are identified and mated to test for germline transmission of the transgene
what does isogenic mean for transgenic mice ?
it means modfiying a piece of DNA within the mouse genome by using DNA that has same/similar homology with that part of the genome to either change it or add or take bits away
how is a transgenic line produced ?
by breeding the chimeric pups until the transgene is contributing to all the cells
what can be done to produce mouse models of human diseases and what is the purpose of this ?
can create mutations in mice that are homologous to disease mutations known to occur in humans
these can be used to as animal models of the disease to aid drug discovery, understand consequence of variation on a homogenous background
what are the different strategies for using transgenics in pharmacogenomics ?
1- ablation of mouse gene
2- transgenic overexpreession of major human allele on wild type background
3- transgenic overexpression of both human alleles on wild type background
4- ablation of mouse gene, then transgenic overexpression of both human alleles
5- concomitant ablation of mouse gene and replacement with both human alleles - this takes longer
what are the challenges associated with transgenic mice ?
identifying ablations from gene
may affect development and cause death
compensatory changes may occur
what does inducible transgenics mean ?
controls the induction of transgene expression - helps to avoid the challenges
- the mouse is allowed to develop normally and then the changes are induced
what are the benefits of inducible transgenics ?
obviates certain problems
- developmental over expression effects such as lethality of knock outs and compensation
what are the various methods for producing inducible transgenics ?
- tet-on, tet-off = the use of a tetracycline responsive promoter- the effect is not induced until you add or remove tetracycline
- cre-lox= this is used for tissue specific knockout- cre recombinase enzyme is able to cut DNA, it cuts out the DNA between lox sites
- combination- tissue specific inducible knock out- this involves knocking out a gene only once youve given or taken away tetracycline
what gene was used to look at variations of metabolism due to different alleles ?
cyp2d6 - 75 different variants
- many environmental factors influence this gene actiivity
why were mice used instead of humans to study the variations in the cyp2d6 gene ?
because variations in other allese contribyte to the cyp2d6 variations so humans are not a good choice to uncover phenotypic consequences whereas mice are genetically homogenous so this reduces the complexity but differences between the ouse and human cyp2d6 isoforms means the mouse is not a good animal moel
why is DEB used as a probe for human cyp2d6 ?
debiquisone is use as a probe because it is hydrolysed by the human cyp2d6 and then the concentration of the product can be measured
rodents cyp2d6 dont hydrolyse the deb
what had to be done to make mice a better model to determine the variation in the cyp2d6 ene ?|
cyp2d6 humanised mice had to be produced
what was the outcome when cyp2d6 humanised mice were made in comparison to wild type mice ?
the humanised mice demonstrated reduced levels of deb compared to wild type because they contains humanised cyp2d6 so they could hydrolyse deb and they also showed greater concentrations of the hydrolysed product of which the wild type showed none
what are beta blockers used for ?
treat high BP and chronic heart failure
why were the efficiousness of beta blockers looked into ?
because humans show a wide range of response to beta blockers
- humans var in enzyme function but also in receptor function
- it was known that there were variations in the beta 1 adrenoreceptr
what are the variations in the beta1 adrenoreceptor ?
it has arginine and glycine variations which alters their response to beta blockers
- the arginine isoform couples with Gs protein more readily
when looking at beta receptor function what was determined when older mice were used /
there was no response to beta adrenoreceptor agonist in the 6 month old mice with arginine in their receptors but the response with glycine receptors was still enhanced
- therefore it would appear that there is some remodelling of the mouse heart
what was seen when acute administration of a beta antagonist was given ?
arginine variant demonstrated much better results - much better block
the glycine variant requires a much higher dose of the beta blocker to induce the block
this means that beta blockers in arginine variants will be more effective
what results were shown when chronic administration of beta blcokers was given ?
this showed that the antagonist was only effective in the arginine variant- it reduced hr significantly but this was not seen in the glycine variant
when clinical trials were observed what was demonstrated with beta blockers ?
individuals genomes were sequences to determiine if they had a glycine or an arginine version
- the beta blockers were much more effective in the patients with the arginine variant
- this demonstrates that this could be a factor involved in why patients effectiveness to beta blockers varies so much
what are epigenetic marks influenced by ?
influenced by the environment - foods, bacteria in your body
what is DNA methylation ?
it doesnt change the genetic sequence
- it causes methylation of cytosines next to guanosines
- this can affect how DNA functions – silence them or enable proteins to bind or not to bind
what does maintence DNMT do ?
it is used to replicate the methylation pattern so they are present on both strands of DNA
What did identical twin studies show about epigenetic marks ?
identical twins at 3 years old showed very similar/identical DNA mehtylation sites which is expected because their genetic is the sam
however identical twins at 50 years old had different DNA methylation
- this provides evidence that the DNA methylation patterns change overtime and it must be due to the environment instead of genetics because identical twins have identical genetic make up
- however you expect twins to be exposed to very similar environemtns and therefore this demonstrates that very subtle differences in enviroment can cause these different epigenetic marks
other than DNA methylation how else can epigenetic marks occur ?
they can also occur on histones - histone modifications
histones have an n-terminal tail which can be modified - phosphorylated or methylation etc - these are more difficult to determine because they are more transient
what are the different terms for transgenic mice ?
transgenic
knock out
knock in
