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used loads of costa ricans to trace the deafness; DFNA1 linked regions: all markers in that regions showed 100% association with the deafness (disease)
-diaphanous gene in flies was really applicable here: because what it affected in flies was applicable to the disorder in humans; encodes the protein important for actin polymerization....huge demand for this in inner hair cells of ear
- why does the gene affect hearing (and not the whole body) if the gene is expressed in all tissues?
- -maybe the ear is more sensitive to loss of function than other tissue?
whole genome sequencing for gene discovery:
here's the family: kids have 2 genetic diseases each, and you have all sequences of both parents and the kids: how do you find the genes?
- 1) get rid of all sequences that are teh same in the individuals: talking about probably closer to 6 mill (identical in all 4 individuals)
- 2) parents have to be heterozygous, children have to be homozygous and MATCH each other
- -but a lot of markers show that inheritance pattern just by chance
- 3) get rid of "normal" changes: like removing polymorphisms that were known to be popular in the population at large, or those that don't change the amino acid sequence (like if the 3rd base pair in a codon is changed, wobble position, same amino acid gets put in the polypeptide)
-still just got down to 2 candidates for each gene
if the gene's you're knocking out are essential, you can detect this because once in teh yeast, one of the 4 offspring spores won't grow
inserting knockout gene into a mouse:
when you stick double stranded DNA into a nucleus, sometimes it recombines hoologously, but sometimes it just goes in randomly
neor is resistance to antibiotics: all constructs will have that: positive selection, only those that survive will have incorporated the construct
but after, on a place with ganciclovir, only the cells that recombined NON-homologously will die, because if the herpes simplex virus kinase is in the genome, the IT will phosphorylate the ganciclovir, subsequently killing it's own cells :(
difference between the first positive then negative selection of cells
two instances where you would just need to inserts a gene into an organism:
- -not necessarily homologously, just in general, the gene/piece of DNA (promoter/coding sequence) needs to be stuck into a genome
- 1) rescue experiment
- 2) reporter construct: basically, enhancer promoter and some sort of marker; don't care where it goes in genome, just htat it gets in
-can do this in a mouse by just inserting foreign DNA into a mouse zygote; gene will show up in progeny
conditional knockouts: Cre recombinase
can knockout genes in certain cells but not all cells (for example, if you needed to study a gene cruicial for ear development, but also embryogenesis...if you knockout too early, embryo will just die)
- causes recombination recombinase; loxP is it target site in the genome
- -loxP have direction; only line up when their directions are the same
the processes it acts in is exactly reversable
in flies and mice, you can cross two animals, one with loxP sites in genome and another with Cre gene ATTACHED to a SPECIFIC promoter (flies: eye color, mice: BRCA1/mammary promoter) to see how the mutation affects the animal, but only in later stages in life