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
