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BB20149 > Transgenic and gene targetting technologies PLS FINSIH > Flashcards

Transgenic and gene targetting technologies PLS FINSIH Flashcards

1
Q

What are the three categories for identifying a gene of interest?

A

Transcriptome profiling, protein-based methods and whole genome sequencing.

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2
Q

What are some of the transcriptome profiling methods?

A

RTase-polymerase chain reaction, DNA microarrays and RNA-seq.

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3
Q

What are two protein based methods to identify a gene of interest?

A

1- and 2- hybrid screening, immunoprecipitation.

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4
Q

What are in vitro limitations of studying gene function compared to in vivo methods, in terms of cells?

A

Not all cells/tissue types are able to undergo in vitro culture.

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5
Q

What are in vitro limitations of studying gene function compared to in vivo methods in terms of matrices?

A

There are limited (heterotypic) matrices and cell interactions.

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6
Q

What are in vitro limitations of studying gene function compared to in vivo methods in terms of factors?

A

Limited/no interstitial, endocrine and other (circulating) factors.

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7
Q

What are in vitro limitations of studying gene function compared to in vivo methods in terms of developmental modelling?

A

There is limited/no developmental modelling.

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8
Q

What are in vitro limitations of studying gene function compared to in vivo methods in terms of organism level analysis?

A

There is none as it’s in vitro - e.g. can’t study learning or response to stress.

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9
Q

What are the steps for manipulating genes in vivo?

A

Manipulation in bacteria (gene cloning), transgenesis, conventional gene targetting and genome editing.

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10
Q

What does transgenesis allow?

A

Visualisation of gene expression in the whole animal.

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11
Q

Give examples of fluorescent reporter proteins.

A

GFP, mCherry.

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12
Q

What is the range of sizes that transgenes can be?

A

0.5kb to >1000kb.

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13
Q

How can transgenesis be carried out?

A

Inject the transgene into the 1-cell embryo (pronuclear zygote) and the gene will integrate into the genome.

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14
Q

What is an alternative of injecting a transgene into an embryo?

A

Microinject the transgene DNA and sperm into an unfertilised oocyte.

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15
Q

What happens in transgenesis when the gene has been injected into the egg?

A

It is transferred to the mother and the embryo gives rise to the offspring. All cells in the offspring have the transgene.

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16
Q

What is the efficiency of injecting the transgene into the egg to produce transgenic offspring?

A

10% will be transgenic.

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17
Q

What are most transgenic animals?

A

Mice.

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18
Q

When were ES cells first reported in mice?

A

1981.

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19
Q

When were ES cells first reported in humans?

A

1998.

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20
Q

Where were the ES cells found in the mice?

A

The inner cell mass of blastocysts.

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21
Q

How can blastocysts be used for gene targeting?

A

Genes can be injected into blastocysts and contribute to development.

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22
Q

What is the method for using ES cell gene targeting?

A

Make a targeting vector DNA construct (TV).

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23
Q

What is the selection method for targeting vectors?

A

Positive-negative selection.

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24
Q

What is required with targeting vectors?

A

Homology arms and a reporter.

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25
Q

What are homology arms?

A

Arms either side of the targeted insertion that are 5-5-15kb in length and recombine by homology direction repair (HDR) with the genome.

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26
Q

What is the typical construct for a transgene?

A

Promoter, gene of interest and then the reporter.

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27
Q

What is a pronucleus?

A

The nucleus of a sperm or egg cell during fertilisation. Sperm becomes a pronucleus after entering the ovum, but before the genetic material fuses.

28
Q

What is pronuclear injection?

A

When the DNA integrates into the genome.

29
Q

Does transgenesis usually result in loss or gain of function?

A

Gain of function, as a gene has been gained.

30
Q

Give an example of positive selection that can be used for the targeting vector.

A

Neomycin resistance, selecting with G418.

31
Q

Give an example of negative selection that can be used for the targeting vector.

A

Thymidine kinase. You can counter select with Ganciclover which is metabolised by thymidine kinase to produce cytotoxin.

32
Q

What is homology directed repair?

A

A mechanism in cells to repair double stranded lesions - used for the homology arm mechanism.

33
Q

What does transfecting mean?

A

Infecting a cell with free nucleic acid.

34
Q

What happens when the targeting vector has been successfully selected?

A

The embryonic stem cells can be transfected with the targeting vector and can be plated in culture flasks - this is hopefully what happens.

35
Q

What is the theory behind positive-negative selection?

A

Positive selection identifies those where the DNA is present, and negative selection is used to identify where the DNA has been inserted CORRECTLY. It allows for fine selection.

36
Q

How does negative selection work in the process of targeting vectors?

A

If the gene is inserted incorrectly (random integration) the thymidine kinase will still be present and the addition of the ganciclover will create a toxin to disrupt the DNA.

37
Q

How can false positives be identified in transgenesis?

A

Genomic PCR.

38
Q

How can the insertion of the correct gene in the correct location be confirmed in transgenesis?

A

Whole genome sequencing (WGS).

39
Q

What happens after the target gene has been successfully inserted into the gene?

A

The ES cells are injected into the blastocyst (3.5 day embryo).

40
Q

What happens when the transgene has been inserted into the blastocyst?

A

It can be injected into a pseudopregnant mother.

41
Q

What is a pseudopregnant mother?

A

Where all the signs of pregnancy are exhibited by an organism, but without a foetus. They make good recipients for transgenesis.

42
Q

How can the correct insertion of the transgene be identified in mice?

A

If a blastocyst is taken from a black mouse and embryonic cells from a white coat mouse, the offspring will have a mixed coat colour.

43
Q

What germ layers do embryonic cells contribute to?

A

They contribute to all germ layers, and sometimes germline cells.

44
Q

What do ES or iPS exist in different species?

A

Humans, rats and pigs.

45
Q

What animals have iPS cells but no ES cells?

A

There are no ES cells for cattle or pig, and iPS cells don’t contribute to germline in pigs.

46
Q

What technique can be used if there are no iPS or ES cells?

A

Embryo genome editing can be used.

47
Q

What are the key factors in genome editing?

A

ZFNs, TALENs and Cas9.

48
Q

What is genome editing?

A

A way of making specific changes to the DNA of a cell or an organism.

49
Q

What is the technology underlying genome editing?

A

Double stranded breaks (dsb) are created at specific places in the genome.

50
Q

How are breaks introduced in genome editing?

A

ZFNs or TALEN (zinc finger nuclease and transcription activator-like effector nuclease).

51
Q

What is ZFN?

A

Zinc finger nuclease

52
Q

WHat are tandem TALE repeats fused to?

A

FokI.

53
Q

What happens after the double-stranded breaks are induced?

A

Cell machinery repairs the break.

54
Q

What is the result of the cell machinery repairing the double stranded break?

A

There may be imprecise repair by non-homologous end joining or precise repair by homology-direct repair.

55
Q

What does non-homologous end joining (NHEJ) result in?

A

Small insertion or deletion.

56
Q

When was the Cas9 system first reported in mammals?

A

2013.

57
Q

Where was the Cas9 system originally from?

A

Bacteria - S.pyogenes.

58
Q

What are the components of the Cas9 system?

A

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA, guide RNA (gRNA), and Cas9 (CRISPR-associated (Cas) helicase nuclease, Cas9.

59
Q

What is the benefit of Cas9?

A

It is simple to use, RNA-guided endonuclease.

60
Q

What is the mechanism behind Cas9-CRISPR?

A

Cas9 introduces a site-specific double strand break, and the cell repairs the break via one of two pathways - the same for ZFNS and TALENs.

61
Q

What are the two pathways in which the dsb can be repaired in the Cas9/CRISPR mechanism?

A

Non-homologous end joining (NHEJ) and homology directed repair (HDR).

62
Q

How do NHEJ ad HDR compare?

A

NHEJ is imprecise as the ends are rejoined, inserting or deleting a few nucleotides whereas HDR is precise as a different DNA molecule is used as a repair template.

63
Q

Where does Cas9/CRISPR work?

A

In mammalian tissue culture cells and embryos.

64
Q

How can genome editing be applied to mammals?

A

A 1-cell embryo zygote can be inected so that all cells in the offspring have the genome edit.

65
Q

What is a modification of the Cas9 pathway?

A

Cas9 nickase fused to reverse transcriptase - it makes a nick not a break, which is safer.

66
Q

What is a modification of guide RNA (gRNA)?

A

gRNA fused to template for RTase called pegRNA (prime editing gRNA). This is one RNA moelcule. This means there is no need for a HDR or HDR template.

BB20149 (16 decks)

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