March 19, 2013.docx

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University of Toronto St. George
Molecular Genetics and Microbiology
Johanna Rommens

Modeling diseases – generating strains that carry mutations that reflect human disease Think about genes itself – high number of genes between mouse and human Methods that have been developed – most sophisticated have been worked through mouse – no limitation being constrained to mouse – but mouse zygote amenable to investigation, moreso than other organisms Gene targeting – generate disease models with precisely change wanted to see in gene – prepare model that has Other models can be more straightforward – knockout particular gene – variety of ways to do that – both processes would use gene targeting – some advancements and some attempts to engineer chromosomes (large scale changes in genome) – want to replicate conditions like Down’s syndrome – large regions spanning that parallel orders of genes for whole chromosomes, many are bunched around where pieces have shifted in evolution Take odd cell out – will not interfere with process of body patterning – can take out some cells, add some cells – manipulate the cells taken out, then put them back – recognize recombinant or gene targeted cells – embryonic stem cells come from blastocyst – in principal, pluripotent – can generate all body cell types – can also lead to generation of germ cells – mouse offspring from these first chimeric mice – can develop strains of mice that carry that particular targeted gene of interest – Variety of mice can be used for this – some strains work better than others – set basis for many models – line called 129 agouti hair is dominant trait – Exploit property of homologous recombination – use this property of cells – not a very common mechanism, so need t make some effort to promote homologous recombination – use endogenous machinery of cells that exist and one can introduce foreign piece of DNA in a very careful way – can recombine with endogenous DNA, achieve desired change – process of homologous recombination is quite rare in mammalian cells in contrast to other organisms that use recombination more frequently Rare in random recombination – Positive-negative selection to enrich for ES cells containing a desired gene-targeting event Gene of interest are red and green portions – wild type gene Can take gene of interest, insert another gene in middle of it, (neo) Incorporate into chromosome DNA – insert linear molecule into DNA – random integration event – in contrast to gene targeting, homologous recombination occurring with favourite gene in which introduce something – neo resistance gene – so this gene renders cell line that takes off this particular fragment of DNA resistant to drug Tk = herpes simplex targeting kinase – enzyme from herpes virus – from herpes virus, some of the nucleotide modifying enzymes can be more promiscuous than human systems or mammalian systems for manipulating nucleotides – cuase cells to die if get processed by tk gene – human cells minimally affected by ganciclovir drug, if incorporate gene targeting construct carefully – if integration – have intact neo gene and intact tk gene – if add neocmycin and ganciclyovir, tk gene will metabolize gancyclovir, resulting in cell death, eliminating random integration – BUT if do gene targeting, using homologous recombination (this is desired), cross over occurs so get gene cross over, get neo resistance gene and the tk gene is lost – so if us ganccylovir selection, then this does not lead to toxic product, because antiviral not processed – cells have neomycin resistance – How used in mice? If one takes mouse – 129 mouse – and take away – take out some inner cell mast cells – and manipulate them – select for gene targeted cells – in presence of neomycin and gancyclovir – in ject into blastocyst – add ES cells to blastocyst – fully viable and contribute to blastocyst into embryo – transplant blastocyst into foster mother and the offspring that had received the cultured ES cells – will then carry – cells carrying genome of that particular ES cells – foster mother is Black6 and the remaining cells in blastocyst are fully Black6 cells – give black mice But 129 – this gives rise to hair that is lighter colour and so offspring that received the injected ES cells will then show the lighter colour and the expectation of blastocyst – chimeric – able to detect chimera by portion of coat colour that is coloured light – get variety of range of degree of coat colour change – select male with most light coat colour – in principal, this should be the mouse that receives the most cells – blastocyst – and can breed male chimera to a female mouse – to a black six mouse and the offspring from these two mice – will now be true heterozygotes – germ cell of male chimera and was the male parent to this particular mouse – and because this mouse – dominant coat colour will be the partent - this________________ – should be able to pass onto offspring – HOW ONE GENERATES MOUSE? Gene targeting approaches Gene knockout generated – inactivate target gene Process of homolgous recombination is process where want in the homolgous recombination process, a fair depth if possible a long length of sequence where there is overlap – want to target – plays off length of homology with whats practical to carry inside plasmid vector – generate din standard plasmids in E. coli – Knock-in – Blue staining throughout skeleton – this gene involved in hedgehodge signalling – promoter left intact by recombination procedure that was used to generate knock-in – can use blue colour looking at embryo – 15.5 day – most mouse have gestation of 18-19 days – expression in jaw, mandible – cleft palate and Gene targeting approaches and strategies – sometimes want to make specific mutations that occur in the disease – recognize that there are knockout alleles and missense alleles – hypomorphs – partial phenotypes – want to generate Site specific recombination – cre-lox – uses for conditional gene inactivation – sometimes, the knockout of some genes is lethal – lethal gene may have role early in development and want to study later stage of disease – end up with dead mouse or embryo (not useful to study) – want to work around that – generate conditional situation – desirable – for many severe genetic diseases (with multiple systems affected) – sometimes, cannot unravel consequence of disease for one organ without another being involved – unable to distinguish them unless can make a model of brain separate from model of other organ – another applicable of coiing up with a system that allows you to look and make conditional model, - knockout of gene of interst only at specific tissues or specific stages of development Scenario – conditional gene inctivaiton using the cre-loxP site-specific recombination Combination of two loxP sites – causes internal deletion – get removal of gene A and end up with genome with single remnant LoxP site but gene of interest removed – dictate where this occurs by how cre recombinase is expressed – Sequence of cre-recombinase and shown for yeast flippase – in similar manner – 34 base pairs and only 34 base pairs – 14 base pairs that flank internal region – internal region is not asymmetric – these sites have direction by virtue of central base
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