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Gene Tech > Week 6 > Flashcards

Week 6 Flashcards

1
Q

Northern Blot/Hybridisation basics

A

ssRNA, electrophoresis, blotting membrane + probe insertion leads to hybridisation

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2
Q

What is RT-PCR?

A

RNA population converted to cDNA by reverse transcription. using reverse transcriptase.

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3
Q

What is the advantage of in situ hybridisation?

A

No assumption about region of gene

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4
Q

What are the 2 disadvantages of in situ hybridisation?

A

Difficult if low expression and is lengthy

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5
Q

What is in situ hybridisation? basics

A

DNA probe put in tissue slides. Blocked (prevent non-specific interaction), incubated and then visualised.

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6
Q

What is microarray and mRNA sequencing?

A

Hybridise oligonucleotides with cDNA sample and know if the cDNA is expressed in specific tissue.

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7
Q

What is microarray and mRNA sequencing used for? (3 points)

A
  • Quantitative data
  • Location and time of expression of gene
  • Address change in gene expression.
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8
Q

How does capture microdissection work? basics

A

Tissue+paraffin on slide. Laser caps around cells and bind to transfer film. Individual cells can then make cDNA.

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9
Q

Promoter enhancer properties? (2)

A
  • Greatly increase transcription rate from promoters on same DNA molecule
  • Function in either orientation
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10
Q

What is the role of the binary system gene expression/ GAL4/UAS system?

A

Used to express transgenes in specific cells.

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11
Q

How does a binary system gene expression system works using GAL4/UAS as an example? basics

A

GAL4: An activator combined with promoter sequence specific to a tissue.
UAS: fused to target gene.
Cross: target gene expressed only in specific tissue. Can find where and when gene is expressed.

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12
Q

Role of activator proteins?

A

Bind to enhancer sequences in DNA and recruit RNA polymerase to bind to the promoter sequence, activating transcription.

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13
Q

What are the benefits of the GAL4/UAS system? basics

A

Easier to cross than build new constructs when multiple targets are to be analysed. In vivo expression + lethal gene study.

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14
Q

What is ideal in a genetic switch? (6)

A
  • Low or no expression when off.
  • High expression when on
  • Fast response
  • Reversible
  • No toxicity or interference in general cell function
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15
Q

How does the tet-off system works? basics

A

No tetracycline: tetA binds to tet repressor, preventing it from binding to tet-o. expression.

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16
Q

How does the tet-on system work? basics

A

Tetracycline: conformational change in tetA, binds to tet-O, expression.

17
Q

What are the uses of transgenic animals? (3)

A
  • Medicine (e.g. model organisms)
  • Agriculture (disease resistance, livestock change, reduce waste)
  • Nutritional supplements and pharmaceuticals (insulin in milk, omega-3 fatty acids in transgenic pigs)
18
Q

How does microinjection work?

A

Micro-inject sperm nucleus, fusion and implantation into female.

19
Q

Problems with microinjection basics

A

Transgene can be inserted anywhere or multiple times in the genome, timing can make it a chimera.

20
Q

How does embryonic stem cell transfer works?

A

Inner cell mass of blastocyst transformed with DNA and induced back into blastocyst. Result: chimera, must cross with wild-type.

21
Q

What is illegitimate recombination?

A

Foreign DNA has an imperfect sequence recognition. Will cleave a gene halting its function, which may be a problem.

22
Q

What is homologous recombination?

A

Perfect sequence recombination, when target gene is flanked by regiosn of homology with host DNA. Replaces native gene with target gene.

23
Q

Positive selection marker

A

Selects for that which HAS the gene. Marker between homology 1 and homology 2.

24
Q

Negative selection markers

A

Counter-select gene, so that it will only be inserted when separate form other markers (illegitimate) and will cause cell death. If legitimate, then negative marker not present and survival occurs.

25
Q

Knockout

A

recombination between two regions of homology substitute part of the gene with engineered gene. Gene of interest is the one already in organism, now null.

26
Q

Knockin

A

Counter-selectable gene outside of homology 1 and homology 2. New gene is placed in place of original gene and is subject to all transcription regulation of the original gene.

Can also have functional gene WITH gene of interest to make a reporter.

27
Q

How does the Cre-LoxP recombinase system works for making a mutation in a specific tissue?

A

Insert gene for Cre recombinase after a promoter. Promoter is specific to a tissue. Therefore, Cre limited to a specific tissue. It then recognises IoxP site.

28
Q

what is the effect of different directions of the recombination?

A

If both forward, then deletion.
If on contrary directions, then inversion.
If IoxP on different chromosomes, then can make large-scale chromosome recombinations.

Gene Tech (12 decks)

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