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Developmental Biology. > Gene Inactivation Techniques. > Flashcards

Gene Inactivation Techniques. Flashcards

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1
Q

What do gene inactivation techniques allow scientists to do?

A

To work out the role of genes.

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2
Q

What are the 2 main ways that we use to knock out genes?

A

By blocking the gene product from getting to the embryo.

Or by eliminating the mRNA that would be translated into the gene product.

Or by removing the gene from the genome.

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3
Q

What does gene inactivation allow us to see?

A

What happens to the organism when that gene is removed.

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4
Q

How does the genetic technique of gene inactivation work?

A

It involves physically removing the gene from the genome.

If there is nothing transcribe then no product will be produced.

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5
Q

Does the genetic technique of gene inactivation work in all species?

A

No.

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6
Q

What happens if a manufactured gene is injected into the pronuclei of a fertilised mouse egg?

A

The new gene will be part of the DNA of the developing mouse and will be expressed.

The fertilised egg will then be inserted into a foster mother and the modified mouse is born.

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7
Q

How can we use stem cells to silence genes?

A

Embryonic stem cells are collected from the inner cell mass and grown in a petri dish.

DNA is then placed into the embryonic stem cells.

The genetically modified cells can be injected into another embryo.

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8
Q

What does the targeting construct do?

A

It manipulates the genome and removes the gene.

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9
Q

The targeting construct must be homologous to what?

A

To sequence of genes on the targeted chromosome.

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10
Q

Why must the targeting sequence be homologous to the chromosome?

A

So that crossover can occur.

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11
Q

The homologous regions on the targeting sequence will be identical to which genes?

A

The genes on either side of the target gene.

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12
Q

How does the targeting sequence remove the target gene?

A

There is an exchange of genetic material between the targeting construct and the target chromosome.

The target gene is given to the targeting construct in exchange for the genetic material on the targeting construct.

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13
Q

What are the selectable markers on the targeting construct?

A

NEOr and thymidine kinase.

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14
Q

What do selectable markers do?

A

They tell us whether targeting has occurred successfully.

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15
Q

What does NEOr do?

A

NEOr gives cells resistance to neomycin.

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16
Q

What does thymidine kinase do?

A

Thymidine kinase will convert an enzyme called gancyclovir into a poison.

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17
Q

What is the most likely outcome of using selectable markers to knock out genes?

A

The targeting construct will not go into the genome or the nucleus and nothing will occur.

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18
Q

What is the 2nd most likely outcome of using selectable markers to knock out genes?

A

The targeting construct will insert itself randomly into the genome of some cells.

When we test the selection markers on these cells none of them will survive.

As the neomycin will kill thise that do not have resistance.

Gancyclovir will kill the rest.

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19
Q

What is the 3rd most likely outcome of using selectable markers to knock out genes?

A

It results in homologous recombination and the only thing that is transferred are the genes that are between the regions of homology.

This means that the cells will survive both neomycin and gancyclovir.

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20
Q

What genes are between the regions of homology in the targeting construct?

A

The neomycin resistant gene and the genes that will be inserted into the chromosome.

The thymidine kinase is not in the region of homology.

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21
Q

What does the conditional knockout technique do?

A

It removes the genes from certain cells in the body and uses the LOX-P system.

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22
Q

What are the 2 components that are involved in the conditional knockout technique?

A

CRE-recombinase.

The LOX-P sequence.

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23
Q

How does CRE-recombinase work?

A

It will recognise the LOX-P sequence in the genome and then cut it out.

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24
Q

What determines how CRE-recombinase works?

A

The orientation of the LOX-P sequence.

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25
Q

What happens if the LOX-P sequences are in the same orientation?

A

CRE will cut the LOX-P sites, remove the piece in between and join the ends back together.

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26
Q

What happens if the LOX-P sequences are facing each other?

A

CRE will cut the piece out and flip it, before re-joining it back together.

This means that the gene that was in that sequence will be backwards and won’t work properly.

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27
Q

What does the CRE-recombinase system allow us to do?

A

To remove a gene from a particular cell but, leave it in all of the other cells.

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28
Q

What must the gene for CRE-recombinase be inserted into?

A

A promoter sequence that will code for a specific expression in a certain cell.

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29
Q

When the CRE-recombinase gene is inserted into the promoter sequence, what will happen?

A

It will be expressed in the area that the promoter sequence usually works in.

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30
Q

Once CRE-recombinase has been expressed by the promoter, what will happen?

A

The gene can be injected into a fertilised egg and CRE will be expressed in the offspring.

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31
Q

The baby mouse that has the CRE gene is called what?

A

The trans activator mouse.

32
Q

What is name of the mouse that the trans activator mouse is mated to?

A

The flox responder mouse.

33
Q

How has the genome of the flox responder mouse been altered?

A

The target gene is genetically modified by adding LOX-P sites on each side using a targeting construct.

34
Q

What happens when the trans activator mouse and loz-p mouse are bred?

A

The offspring has only the target gene removed by CRE-recombinase.

35
Q

What happens to the cells that do not have CRE-recombinase?

A

They will express the target gene.

36
Q

How does the antisense technique work?

A

mRNA is stopped from binding to a ribosome and being translated.

37
Q

What prevents the mRNA from being translated into a protein in the antisense technique?

A

Antisense oligonucleotides.

38
Q

Is the antisense technique permanent?

A

No. It only lasts for a short time.

39
Q

What DNAs does the antisense technique use?

A

Very short, single stranded DNA’s that have a complementary sequence to the mRNA target.

40
Q

What is the very short DNA strand in the antisense technique called?

A

The antisense strand.

41
Q

What does the antisense strand do in the antisense technique?

A

It will bind to the mRNA and stop it from being translated by a ribosome.

42
Q

Where does the ribosome bind to the mRNA?

A

At the 5 prime end.

43
Q

How do we get 100% inhibition of the genes in the antisense technique?

A

If binding occurs at the AUG triplet or the 5 prime end of the mRNA.

44
Q

If the antisense strand is placed on a different region of DNA to the 5 prime end or the AUG triplet will there be protein synthesis?

A

Yes.

45
Q

When mRNA is originally synthesised, what will it contain?

A

Introns and exons.

46
Q

What happens to the introns in mRNA?

A

They are spliced out.

47
Q

What is alternative splicing?

A

When an exon is removed.

48
Q

What is formed by alternative splicing?

A

Alternative proteins.

49
Q

How can we genetically modify an organism using alternative splicing?

A

By binding an antisense strand to the splice site.

50
Q

How does alternative splicing take place?

A

If an mRNA has 2 exons and 3 introns and alternative splicing takes place then exon 2 may be removed and only 1 exon is used leading to the formation of a different protein.

51
Q

How can we remove an exon from a DNA strand?

A

If an antisense strand is bound to an exon that will lead to the formation of a harmful protein it will eliminate its formation.

52
Q

What is the SIRNA technique?

A

It is similar to the antisense technique.

SiRNA’s will bind to the mRNA and degrade it.

This means there is nothing for the ribosome to translate.

53
Q

What method of genetic manipulation is used for gene therapy?

A

The SIRNA technique.

54
Q

What cells will the SiRNA technique work in?

A

Only in certain cell types.

55
Q

What kind of genetic materials are introduced into the embryo in the siRNA technique?

A

Small double stranded RNA or long double stranded RNA molecules.

56
Q

What are the SiRNAs also known as?

A

Micro RNAs.

57
Q

How are the SiRNAs synthesised?

A

They can be synthesised naturally.

For genetic manipulation they are synthesised artificially.

58
Q

Micro mRNAs must be homologous to what in the SiRNA technique?

A

They must be complimentary to the targeted mRNA.

59
Q

What happens to the long strands of SiRNA once they enter the target cell?

A

A protein called DICER will cut it up.

60
Q

What does DICER cut the SiRNA into?

A

20-25 BP double stranded RNAs.

These are the micro RNAs.

61
Q

What happens to the micro RNAs once they have been cut up by DICER?

A

They will then bind to a protein complex called RISC which will remove 1 strand of the micro RNA.

62
Q

What does RISC stand for?

A

RNA Induced Silencing Complex.

63
Q

When is the RISC protein active?

A

When it is bound to the single strand of RNA.

64
Q

What happens once the RISC protein is active?

A

It is directed by the single stranded micro RNA to a complimentary target strand which the RISC complex will cut up.

65
Q

How do neutralising antibodies work?

A

They bind to specific proteins and block the active site or change the shape of the protein.

They can be used to target cell surface receptors which often form as dimers (2 proteins that come together).

The antibody will target the dimer and block dimerisation and the receptor cannot be formed.

66
Q

What is dominant negative technique?

A

It is used when there are many proteins that come together to form an active complex.

Scientists will generate one of the components of the active complex in a mutated form.

When it binds to the other proteins to form the complex, it will cause the complex to be dis-functional.

67
Q

How does the dominant negative technique work in dimers?

A

If a faulty dimer is used, then the receptor will not be able to transmit the message from a ligand to the nucleus and no protein will be translated.

68
Q

The antisense technique uses what kind of genetic material?

A

Single stranded DNA oligonucleotides of varying length.

69
Q

The SiRNA technique uses what kind of genetic material?

A

Large dsRNA or small (21-25 bp) SiRNAs.

70
Q

What cellular machinery does the antisense technique need?

A

Does not depend on any cellular organelles.

71
Q

What cellular machinery does the SiRNA technique need?

A

Depends on DICER, RISC and RdRP.

72
Q

How efficient is the antisense technique?

A

Not efficient.

73
Q

How efficient is the SiRNA technique?

A

Depends on the cellular machinery.

74
Q

Which species is the antisense technique effective in?

A

All species.

75
Q

Which species is the SiRNA technique effective in?

A

It is limited to particular species.

Developmental Biology. (21 decks)

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