In vivo modification of gene expression in eukaryotes Flashcards

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1
Q

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

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

How can you induce random, untagged mutations?

A
  1. Radiation
  2. Chemically
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3
Q

What’s forward genetics?

A

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

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4
Q

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

A

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

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5
Q

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

A

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.
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6
Q

What can TALENs do, that CRISPR cant?

A

TALENs can modify mitochondrial DNA.

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7
Q

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

A

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).

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8
Q

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

A

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

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9
Q

What’s a gene drive?

A

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.

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10
Q

Give to expamples of post-transcriptiona lgene eilencing.

A

Antisense RNA
RNAi

(Mechanisms are discussed in another lecture).

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10
Q

Give to expamples of post-transcriptionalc gene eilencing.

A

Antisense RNA
RNAi

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11
Q

Describe the RNAi mechanism.

A

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.

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12
Q

What types of short RNAs can guide the RISC complex?

A
  1. miRNAs (endogenous hairpin –> 2xmiRNAs)
  2. siRNAs (from long dsRNAs)
  3. shRNAs (dsHairpin RNA, left- or right sided) (exogenous RNA that mimics mRNA9.
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13
Q

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

A

miRNA: represses translation, promotes RNA degradation.

siRNA: cleaves RNA

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

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14
Q

Why does RISC denaturate dsRNA?

A

Virus-fellers.

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15
Q

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

A

20-25 nt, 2-3 nt 3’ overhang.

16
Q

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

A

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.

17
Q

Can you overexpress a gene too much?

A

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

18
Q

What’s the role of miRNA in eukaryotic genomes?

A

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

19
Q

What are shRNAs?

A

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.

20
Q

What’s TGS? Why is TGS important?

A

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.