GV8: Stem Cells & Gene Manipulation Flashcards
what are the three main approaches to gene knockdown in cell models
RNA interference, antisense, CRISPR
how does antisense work
single stranded RNA/DNA of 15-30 nucleotides that is complementary to the target DNA
how does siRNA-mediated RNA interference work
injected single stranded complementary RNA (antisense) should hybridise to target mRNA and induce mRNA knockdown
double stranded mRNA is more effective at inhibition than antisense
what mediates RNA interference
RNA interference silencing complex
doublee stranded RNA targeting coding regions of mRNA
how does CRISPR gene editing work
insertion of viral DNA fragments into bacterial genome; detection and destruction of virus upon re-infection; CRISPR region transcribed into primary transcript and tracrRNA, cleaved to produce crRNA which is complementary to a specific virus, Cas9 cleaves viral DNA and destroys the virus
what are oligonucleotides
(antisense, siRNA, gRnA)
large, negatively charged molecules
how are oligonucleotides delivered
vesicles, attachment of delivery vehicles, viral delivery, electroporation
how are oligonucleotides packaged into vesicles
with cationic lipid and polymers
mix with transfection reagent, add to suspended cells, adherent cells layer transfection reagent on bottom of plate then add cells (negative transfection)
what are the issues with olglinucleotides packaged into vesicles
toxicity, poor transfection of primary cells
how does electroporation work
expose cells to electric field that produces pores in the membrane; extensively employed with primary cells
what are the issues with electroporation
can destroy cells
what are the advantages of using antisense, siRNAs and CRISPR
only require knowledge of the mRNA sequence, can get potent knockdown of mRNA, cheap and easy to identify effective sequences, RNAi and CRISPRi is amenable to high throughput library screening
what are the disadvantaged of using antisense, siRNAs and CRISPR
delivery
