In vivo modification of gene expression in eukaryotes Flashcards
There are three categories of knock-out mutations, which?
- Random, untagged (indels)
- Random/selected, tagged (transposons, T-DNA transfer)
- Directed (CRISPR, TALENs, ZFNs)
How can you induce random, untagged mutations?
- Radiation
- Chemically
What’s forward genetics?
It’s when you induce genomic change and screen for a certain phenotype.
How can you prepare an insert for homologous recombination in a target cell?
Use PCR to amplify the gene with primers that have overhangs hosting homologous domkains for wherever you want it to be inserted.
What does RIP stand for? What’s the mechanism behind it?
RIP = Repeat-induced point mutation.
- Gene becomes point mutated the same way in both alleles in parent X.
- During meiosis, both of the alleles from parent X will become further mutated into loss-of-function + methylated.
- One allele from parent y is paired with one defect allele from parent x.
- Parent x allele is recessive –> function is retained from allele Y.
What can TALENs do, that CRISPR cant?
TALENs can modify mitochondrial DNA.
How can the CRISPR/cas9 system be used for DNA editing?
Cas9 can be conjugated to an RNA-editing enzyme, which can induce substitution-mutations (ex C –> U), which will be repaired to T (C–>U–>T).
When would you NOT want a strong promoter when overexpressing a gene?
When you’re studying sensitive genes which take part in sensitive mechanisms.
What’s a gene drive?
A gene drive is when genes are engineered to be propagated to a smaller extent than it would normally throughout a population. This may be done in malaria-carrying mosquitos, the genes required for malaria to attach may be removed.
A gene drive is based on that cas9 is constiuitively incorporated with a gRNA that cleaves all alleles that don’t carry a certain resistance gene. The allele will be repaired by homologous recombination, and then hosts the gene.
Give to expamples of post-transcriptiona lgene eilencing.
Antisense RNA
RNAi
(Mechanisms are discussed in another lecture).
Give to expamples of post-transcriptionalc gene eilencing.
Antisense RNA
RNAi
Describe the RNAi mechanism.
1.dsRNA is present in the cell (either exogenous or endogenous).
2. Dicer cleaves dsRNA into smaller fragments (miRNA/siRNA).
3. RISC denaturates dsRNA –> ssRNA (siRNA) and binds a strand which is used for guiding.
4. siRNA/miRNA/shRNA guides RISC-complex to complementary mRNA.
5. RISC complex acts differently based on what RNA is guiding it.
miRNA: Inhibition and degradation
siRNA: Cleavage
rasiRNA (deriving from repeated sequences): Inhibits the mRNA by sterically blocking polymerases.
What types of short RNAs can guide the RISC complex?
- miRNAs (endogenous hairpin –> 2xmiRNAs)
- siRNAs (from long dsRNAs)
- shRNAs (dsHairpin RNA, left- or right sided) (exogenous RNA that mimics mRNA9.
Describe the differences in how miRNA, siRNA and rasiRNA (in the RISC complex) silences transcription.
miRNA: represses translation, promotes RNA degradation.
siRNA: cleaves RNA
rasiRNA (derives from repeat-sequences): Downregulates transcription by binding the mRNA.
Why does RISC denaturate dsRNA?
Virus-fellers.
