Gene Manipulation

Question 1

Indels

Answer 1

• inserts / deletions
• Most likely induces Frameshifts
• Most often leads to truncated proteins ( “Knock-out”)

Question 2

Over-expressing genes

Answer 2

The GAL4 / UAS system
enables ectopic (additional)
expression of a gene

Question 3

Reducing gene expression

Answer 3

RNA interference (RNAi) is natural process in eukaryotes utilised to:
* Defend against viruses
* Destroy Transposable Elements (LINEs & SINEs)
* Fine-tune gene expression temporally
Two pathways:
1. Degrades foreign dsRNA into short interfering RNA (siRNA) = Defence
2. Systhesises endogenous micro RNA (miRNA) using Drosha = Regulation

Dicer degrades dsRNA into smaller (~20bp) RNA fragments (siRNA or miRNA)
These interact with RNA Interacting Silencing Complex (RISC) to inhibit gene
expression

If siRNA or miRNA perfectly matches mRNA it’s targeting  degradation
If not, siRNA / miRNA bulges  translation suppressed
* recruits proteins to bind 5’-cap of mRNA and inhibits ribosome binding

Question 4

RNAi Limitations

Answer 4

• miRNAs can have off-target effects, knocking-down other genes
• Different miRNAs have different efficiencies so knock-down levels vary