The functional genome Flashcards
what does whole genome sequencing do
rapid modern method for high throughput DNA sequencing
captures sequence of coding region of genome
cell culture techniques (in vitro)
removal of cells from an animal and growth in favourable conditions
primary cells have finite divisions but can immortalised to provide continous source
provides cheap, rapid and reproducible model for studying normal physiology and biochemistry of cells
alternative = reduce no. of animals being used in research, less restrictions
many tissue specific cell lines commercially available
cell culture: gene knockdown
short hairpin RNA
based on endogenous microRNA gene silencing
modified to include GOI complimentary sequence
packaged in DNA plasmid, expression controlled by RNA polymerase III promoter
50-70nt, exits nucelus, cleaved by nuclease aka dicer
cleaved segments bind to ran induced silencing complex and direct cleabage and degradation of complementary mRNA
short interfering RNA
where is GOI’s encoded protein localised
antibody staining = protein of interest, downstream target
transfect cells with GFP tagged GOI
transfect cells with GFP tagged mutated GOI
why isn’t cell culture enough
cells behave diff in Petri dish
don’t stimulate actual conditions in organism
no info about gene expression and function,
mouse = mammalian model for human genetic disease
similar to humans
precise mutation to recreate disease
small, reproduce quickly, easy to handle and transport
accelerated lifespan
making a mutant mouse
targeting vector constructed and introduced to nucleus of pluripotent ES cells
HR integrates in cassette, ES selected, based on AB
positive ES cells grown to blastocysts and implanted to pseudopregnant recipient mice
zebrafish gene knockdown
a. translation blocking MOS
b. splicing inhibiting mos
MOs block gene specific translation or splicing
CRISPR/Cas 9 system
clustered regularly interspaced short palindromic repeats
CRISPR associated protein 9
bacterial adaptive immune system
protospacer
guide RNA binds to strand of genomic DNA, cas9 endonuclease binds to non protospacer portion of gRNA and PAM of dna
mutations can be introduced through NHEJ and HDR
