TB8 Flashcards
Define: hypomorphic phenotype
The gene isn’t knocked out completely
Why is a hypomorphic phenotype often useful?
It allows for the study of phenotypes without having a complete knockout phenotype which may arise in death of the cell/organism.
Define: allelic series
A collection of different mutants with slightly different defects.
(This gradation can also be achieved using RNAi by differing the concentration of siRNAs.)
Define: trans gene
Foreign/lab-produced DNA
Define: RNAi
An umbrella term to describe homology-dependent gene silencing events triggered by dsRNA
Define: Dicer
An RNase II family member that produces siRNAs
Define: RISC
RNA-induced silencing complex. A multicomponent machine that enforces silencing. Contains an RNA binding protein of the Agonaute family.
List 3 sources of ds-RNA that can be used in RNAi
- Injection in vitro
- Digestion of dsRNA synthesized in bacteria
- Viral dsRNA
(transgene dsRNA, transposon dsRNA)
Define: microRNA
Genomically encoded non-coding RNA that helps regulate gene expression, particularly during development.
Compare miRNA to siRNA
Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but miRNAs must first undergo extensive PTM
Define: gonad
An organ that produces gametes
How do you transform bacteria to express dsRNA of interest?
A plasmid is inserted that contains the gene of interest, as well as an IPTG-inducible bacterial promoter at each end. This will produce transcripts in both directions so you end up with dsRNA, rather than ssRNA.
Suggest additional controls for an RNAi experiment
You could test to see whether the RNAi was actually successful and what the levels of knockdown within each worm was by testing for the protein/mRNA levels of air-1.
You could also use a western blot to see how much air-1 was produced.
Reintroducing the WT air-1 gene to rescue the phenotype would tell you whether the gene responsible for these mutations is actually air-1 or some other mutation (unlikely to be anything else, but could be worth testing for as spontaneous mutations can occur).
Why might there be differences between an RNAi knockout worm, and a mutant strain worm?
The different phenotypes observed may be because RNAi only hits the maternal contribution, but the mutant strain doesn’t. RNAi also works much less in neurons as they don’t have the uptake mechanism to uptake the dsRNA and so are somewhat protected.
Define: off-target effect
If a target sequence appears more than once within a genome (or large portions of the sequence), there’s a chance that Cas9 will be guided to a non-target location that has the complementary sequence to the guide RNA. This will result in Cas9 cutting ‘off-target’ and can arise in genetic mutations.
