RNAi Flashcards
What is RNAi?
- RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Historically, RNA interference was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling.
What is the Basic RNAi mechanism?
What is Dicing?
- Dicer: the RNAseIII enzyme that cleaves the trigger dsRNA into short pieces.
- Dicer is like a ruler that can measure off ~25 nt off the end of a dsRNA molecule.
- These small chopped up RNA bits are called siRNAs (for small interfering RNAs)
What are the ways to deliver RNAi?
- Injection Delivery of RNAi
- ASOs!
- Transfection (as “free” dsRNA)
- mammalian cells
- Transformation (integrated as a DNA transgene engineered to produce a dsRNA)
- Transgenic
- Soaking/feeding!
- (C.elegans)
- Bacteria
Is RNAi found in bacteria?
nah
How can we use RNAi as a tool?
- Gene knock out/down with no genetics!
- Design and generate double stranded RNA
- short hairpin RNAs (shRNAs) necessary for mammalian systems because of the interferon response; longer sequences OK for lower eukaryotes
- interferon response leads to apoptosis
- Deliver dsRNAs or shRNAs to organism/cell
- Verify that protein is knocked down
- Western Blots
What is slicing?
- Slicer: this enzyme cuts the target mRNA.
- Slicer AKA “Argonaute” proteins, with characteristic domains (PAZ, PIWI).
- The PIWI domain resembles a similar domain in RNaseH, an enzyme that cuts RNA:DNA hybrids.
What is a real-world application of RNAi?
Application: Study the genetics of tumour suppression mediated by p53
What is p53?
tumour suppressor
How would you transfect a mouse with RNAi?
***Use viral vector (maybe) to deliver RNAi
***
What test is this?
WWWWWEEEESSSSTTEEEERRRRNNNNNN
What would the GFP control for?
It is not native so the presence of it would mean the gene was NOT knocked down
What is the vector lane?
What p53 is most effective?
Probably empty or nothing of relevance.
Shows vector is not interfering with the experiment
C is the most effective
How is RNAi structured?
Why would you avoid the conserved region?
So you don’t knock down fucking everything that is similar (like all kinases)
What is the easiest way to make ASOs?
Pay someone else
What is the surveillance pathway of RNAi?
What is the micro RNA pathway?
Describe the biogenesis of micro RNA
What is RNAi being used to knockdown?
The facking exosome
