Genetics Toolbox 2

Question 1

How can you manipulate the structure of genes?

Answer 1

Gene duplication
Gene deletion
Single point mutations
Generation of Indels

Question 2

What are indels?

Answer 2

Insertions and deletions are common forms of gene manipulation
Most likely induce frameshift
Leads to truncated proteins - knock out

Question 3

How do you change the gene’s structure and expression?

Answer 3

Changing the gene’s structure - altering the product
Changing the gene’s expression - altering the amount

Question 4

Describe the CRISPR revolution.

Answer 4

Provides natural viral defence in bacteria
crRNA contains spacer
tracrRNA stabilises crRNA within Cas9 protein
tracrRNA + crRNA + linker = single guide RNA (sgRNA)
Advantages - very precise and easy to engineer

Question 5

How can you manipulate gene expression?

Answer 5

Transgenic amplification - ectopic gene expression (knock in)
RNA interference - gene knock-down
The GAL4 / UAS system enables ectopic (additional) expression of a gene

Question 6

What are the 2 pathways used to reduce gene expression?

Answer 6

Defence - degrades foreign dsDNA into short interfering RNA
Regulation - synthesis endogenous micro RNA using Drosha

Question 7

What is RNAi ?

Answer 7

Dicer degrades dsRNA into smaller RNA fragments
These interact with RNA Interacting Silencing Complex to inhibit gene expression

Question 8

Describe the effects of perfect/ imperfect match of RNAi

Answer 8

If siRNA or miRNA perfectly matches mRNA it’s targeting - degradation
If not, siRNA / miRNA bulges - translation suppressed


Question 9

What are the RNAi limitations?

Answer 9

miRNAs can have off-target effects
Different miRNAs have different efficiencies so knock-down levels vary