In vivo modification of gene expression in eukaryotes

Question 1

There are three categories of knock-out mutations, which?

Answer 1

1. Random, untagged (indels)
2. Random/selected, tagged (transposons, T-DNA transfer)
3. Directed (CRISPR, TALENs, ZFNs)

Question 2

How can you induce random, untagged mutations?

Answer 2

1. Radiation
2. Chemically

Question 3

What’s forward genetics?

Answer 3

It’s when you induce genomic change and screen for a certain phenotype.

Question 4

How can you prepare an insert for homologous recombination in a target cell?

Answer 4

Use PCR to amplify the gene with primers that have overhangs hosting homologous domkains for wherever you want it to be inserted.

Question 5

What does RIP stand for? What’s the mechanism behind it?

Answer 5

RIP = Repeat-induced point mutation.

1. Gene becomes point mutated the same way in both alleles in parent X.
2. During meiosis, both of the alleles from parent X will become further mutated into loss-of-function + methylated.
3. One allele from parent y is paired with one defect allele from parent x.
4. Parent x allele is recessive –> function is retained from allele Y.

Question 6

What can TALENs do, that CRISPR cant?

Answer 6

TALENs can modify mitochondrial DNA.

Question 7

How can the CRISPR/cas9 system be used for DNA editing?

Answer 7

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).

Question 8

When would you NOT want a strong promoter when overexpressing a gene?

Answer 8

When you’re studying sensitive genes which take part in sensitive mechanisms.

Question 9

What’s a gene drive?

Answer 9

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.

Question 10

Give to expamples of post-transcriptiona lgene eilencing.

Answer 10

Antisense RNA
RNAi

(Mechanisms are discussed in another lecture).

Question 10

Give to expamples of post-transcriptionalc gene eilencing.

Answer 10

Antisense RNA
RNAi

Question 11

Describe the RNAi mechanism.

Answer 11

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.

Question 12

What types of short RNAs can guide the RISC complex?

Answer 12

1. miRNAs (endogenous hairpin –> 2xmiRNAs)
2. siRNAs (from long dsRNAs)
3. shRNAs (dsHairpin RNA, left- or right sided) (exogenous RNA that mimics mRNA9.

Question 13

Describe the differences in how miRNA, siRNA and rasiRNA (in the RISC complex) silences transcription.

Answer 13

miRNA: represses translation, promotes RNA degradation.

siRNA: cleaves RNA

rasiRNA (derives from repeat-sequences): Downregulates transcription by binding the mRNA.

Question 14

Why does RISC denaturate dsRNA?

Answer 14

Virus-fellers.

Question 15

What characteristics will the siRNA have after being cut from dsRNA by dicer?

Answer 15

20-25 nt, 2-3 nt 3’ overhang.

Question 16

Explain how one siRNA silencing can lead to systematic mRNA silencing.

Answer 16

The siRNA amplification loop. siRNA may bind to an mRNA (w/o RISC complex). RNA-depend RNA-polymerase may then use the siRNA as a primer and generate a long dsRNA sequence from the mRNA template. The long ds-mRNA gets cut by the RISC-complex, generating more siRNA.

Question 17

Can you overexpress a gene too much?

Answer 17

Yes. It causes gene silencing. It’s mechanism to protect the genome from viral vectors and transposons.

Question 18

What’s the role of miRNA in eukaryotic genomes?

Answer 18

There’s hundreds of miRNas per genome, they derive from gene sequences and act in a coordinated fashion. miRNAs are central for cell development.

Question 19

What are shRNAs?

Answer 19

shRNAs are short dsRNAs which don’t get degenerated by the RISC complex. There are left- and right-sided shRNAs which can be incorporated into the RISC complex.

Question 20

What’s TGS? Why is TGS important?

Answer 20

TGS = Transcriptional gene silencing.

If TGS is a consequence of siRNA/dsRNA, the consequences last multiple generations.

When TGS is downregulated, transposon activity is increased (the genome stability is reduced). Heterochromatin formation is also disrupted if a cell is unabale to perform TGS.