Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Intercal - Advanced molecules, membranes and cells > Genetic Manipulation Technology 3&4 > Flashcards

Genetic Manipulation Technology 3&4 Flashcards

You may prefer our related Brainscape-certified flashcards:
Organic Chemistry 101 flashcards
1
Q

Define transcription

A

Transcription is the process of turning DNA into mRNA by RNA polymerase II

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What two components are required by RNA polymerase to start transcription

A

Enhancers
Core promotors

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Define enhancers

A

Enhancers required to increase RNA polymerase II activity at the promoter.

Some cells require more than one enhancer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Give some characteristics of enhancers

A

Variable distances from the start of the gene
Can work on more than one gene
Dont produce protein product
Indicate when, where and how much of a gene is sequenced.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the use of enhancers in genetic cloning

A

Can be isolated and placed next to the gene in genetic cloning

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Define promotors

A

Promoters are DNA sequence immediately next to transcriptional start site of the gene coding region (exons).
Found at the pre-initiation complex (PIC) - RNA polymerase etc

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Can promoters transcribe on their own

A

Usually not, need enhancers to bring DNA binding transactivators

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Once the transcribed mRNA is formed, what is left

A

An mRNA sequence with introns and exons (coding regions)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What happens to this mRNA sequence before translation

A

Capping at 5” end to stop degredation
Poly A tail at 3” end
splicing of introns - different splicing combos make different genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Define translation

A

The process of changing a spliced mRNA sequence into protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How does translation work

A

spliced RNA enters ribosome and is paired with tRNA equivalent which starts and continues a chain of amino acids

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is a reading frame

A

A stretch of successively arranged codons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is an open reading frame

A

a reading frame without a Termination Codon in more that 50 codons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Define a transgenic animal

A

An animal which can stably contain an exogenous gene that has been artificially inserted.

These genes can usually be inherited.
All cells contain this gene (inserted embryonically)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How to build a transgene - 5 stages

A

Isolate the gene and form cDNA (gene with no introns) - easier to handle as smaller

Ensure promotor sequence before gene and regulatory sequences (transcription factors)

Ensure correct RNA processing - ensures poly A tail

Genetically ligate an intron as mRNA processing works better with an intron present

Termination sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Why is it difficult to get transgenes into cells

A

Transgenes are hydrophilic while cell membranes are hydrophobic

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Give 4 ways how transgenes enter cells

A

Direct micropipette injection

Electroporation - shocks cells to make a hole to let DNA in

Chemical transfection - Incubate cells in a culture medium containing DNA and a chemical that wraps the DNA up diffuses through the cell membrane meaning the transgene can enter the original DNA

Infection - expose cells to viruses carrying transgene DNA that will infect cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How to make all cells of an animal have the transgene

A

Enter into one cell embryo

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What happens if the transgene is added to a multicell embryo

A

Mosaic mouse (some cells have it some not)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What is the most efficient way to enter a transgene into a one-cell zygote

A

Direct injection with a micropipette then insert back into animal

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What are some issues with direct injection

A

Relies on the DNA replair enzymes integrating the DNA

Often the transgene will integrate as a concatemer (multiple copies of the same gene in a row) - we have no control of how much is copied.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Give some issues of transgenic animals

A

Weak promoter

Excess copy number

Position of integration into genome - can get expressed in the wrong organ

Epigenetic modification - if repeat is too big it is shut down usually

Genetic background of animal used as the subject

Not all the subjects will be expressing then gene or even carrying it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What type of transgenes work best

A

Very big transgenes where the promoter is right at the start usually work best

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is viral-mediated gene transfer

A

Using viruses to get transgenes into cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

How do retroviruses work

A

Infect cells - RNA gene is reverse transcribed into DNA and enters host genome

Virus DNA contains all the genes for proteins needed to make a more infective virus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What is the gag gene of a retrovirus

A

Encodes proteins of nucleprotein core of viron

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

What is the pol gene of a retrovirus

A

Encodes reverse transcriptase and integrase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is the env gene

A

Encodes surface proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

What is a packaging cell

A

A cell which has the gag, pol and env genes to make the viral particles

30
Q

What is the purpose of packaging cells

A

Allows the transgene to enter with a packaging signal and all the other viral proteins are formed –> make viral subunits with the transgene in them and burst out the cell –> this is collected and used to infect other cells

31
Q

Why are viruses not usually used to create transgenic animals where the gene is found in every cell

A

Embryos are usually able to spot and silence viral sequences by methylating them

32
Q

When are viruses used in transgenic animals

A

Usually adults or mid-gestation embryos therefore creates a mosaic

33
Q

What 3 other viruses types are used

A

Lentiviruses (retroviruses type)
Adeno-associated virus AAV
Adenoviruses

34
Q

Features of lentiviruses

A

HIV derived and very efficient and stable for long term expression of transgene

35
Q

Features of adenoviruses

A

DNA genomes that are large
Short-term (few weeks until killed by subjects immune system)
Dont integrate

36
Q

Features of adeno-associated viruses AAV

A

Much smaller than AV
Can infect non-dividing cells and can integrate so popular for gene therapy

37
Q

Why are viruses good for transgenes and name 2 downsides

A

Can infect all organisms but may only work on dividing cells and can also be silenced
Can mutate and spread

38
Q

Why are conditional gene/tissue specific knockouts done

A

Sometimes when genes are knocked out the organism doesnt survive long enough to determine the true function of that gene. Therefore the organism must be left to reach gestation and the gene must be knocked out there after

39
Q

Difference between homologous recombination and tissue specific knockout

A

Homologous recombination - knockout of all genes as embyro

Tissue-specific knockout - after gestation

40
Q

What is cre-lox technology

A

Cre is a site speficic recombinase enzyme because it recognises LoxP (34 base pair DNA sequence)

Used in vivo to integrate bacteria DNA

41
Q

How is cre-lox technology used in kockouts

A

Between two LoxP sites the cre removes that genetic sequence

42
Q

What is a floxed gene

A

A gene that has loxP sites either side so cre can remove it

43
Q

How is a tissue-specific knockout carried out

A

One mouse with a floxed gene of interest (LoxP either side)

One mouse with a cre recombinase enzyme in tissue-specific site (e.g. pancreas)

Allow them to breed –> gene is knocked out in ONLY the tissues where cre is expressed

44
Q

How is a floxed gene formed?

A

Homologous recombination in embryonic stem cells (using targeting vector and abx resistance marker)

Positive negative selection takes place and sequence surviving cells to make sure only floxed genes are found.

Inject floxed genes into blastocysts then get rid of abx resistance gene as can interfere with floxed gene.

45
Q

How is the neomycin resistance (Abx resistance marker) gene removed

A

Breed the mice with transgene and the abx resistance marker will be knocked out due to the floxed site being found before neomycin site
-> will now get flexed allele

46
Q

What is difficult about using the cre-loxP construct

A

Finding a robust promoter which is strongly pro-cre expression only- it is the rate limiting step

47
Q

How would you control when a gene is knocked out in a subjects lifetime

A

The expression of cre can be controlled in a timely matter in 2 ways -
Tamoxifen
Tetracycline

48
Q

How does tetracycline cause cre knock in at controlled time

A

In normal rat…
-The Tet-activator protein ⟶ Binds to the tet operon (tetO) to activate transcription of Cre
-BUT not in the presence of tetracycline (as this abx will bind to tetA)

SOOOOO……..
-if you keep mice dosed with tetracycline
-> the tetracycline binds to tet activator protein (to stop it working.)

When tetracycline removed…
-> TetA binds tetO
-> Cre is transcribed
-> floxed gene knocked out

summary:
If keep mice dosed with tetracycline - binds to tet activator –> The cre gene isnt turned on.
so the mice survives until the mice isnt given tetracycline anymore.
- allows the cre gene to be activated and knocked in replacing the floxed gene

tet-off system:
tet A protein bend to tet O to activate cre transcription when no tetracycline, tetracycline blocks this

49
Q

How does tamoxifen induce cre expression at a controlled time

A

Cre-ER (Cre with an eostrogen receptor) is used to allow it to bind to tamoxifen, when this occurs, cre-mediated recombination is carried out

50
Q

Features of Cre LoxP

A

Established
Not 100% efficient
Not reversible

51
Q

Features of tetracycline/tamoxifen inducible systems

A

Sometimes routine
Efficient
Reversible

52
Q

Why are adenoviruses good for gene transfer

EXTRA READING - LEE

A

Easiest viral transfer - most humans have an adenovirus receptor and integrin receptor meaning the virus can easily invade humans and produce high levels of transgene.

Gutless adnoviruses - less of an immune response by humans so last longer to make transgene

A lot of research now has established safe doses and routes

Doesnt integrate into host genome so stops any mutagenesis therefore increasing successful expression of transgene

53
Q

Give an example of adenoviruses being used to treat conditions

A

1992 - Alpha-1 antitrypsin given to a person with alpha-1-antitrypsin deficiency via adenovirus into the person hepatocytes

54
Q

How have adenoviruses been used as anticancer agents (gendicine)

EXTRA READING - LEE

A

Gendicine is a recombinant adenovirus engineered to express wildtype-p53 (rAd-p53) in tumour cells. This virus is designed to treat patients with tumors which have mutated p53 genes.

It also blocks VEGF in tumour cells preventing it from growing

First ever gene therapy

55
Q

What is rosa26 good for

Hoinstein et al. 2008

A

Prevents transgene instability and therefore epigenic modification through methylation etc.
Over 130 knock-in lines have been created based on the ROSA26 locus.
A human homolog has been identified of the ROSA26 locus - a site that can be used for genetic therapy in humans.

56
Q

Why are viruses poor choices for making a fully transgenic animal

A

Because of long-term silencing of viral gene in eukaryotic cells

57
Q

How does Forni at al. 2006 give an example of tamoxifen-induced cre-loxP knockout

A

Uses cre-loxP tamoxifen to show how high levels of cre cause hydrocephalus in mice.
The cre mice and loxP mice were allowed to mate to show how cre causes microcephaly and hydrocephalus by having low levels of neural progenitor cell proliferation and high levels of cell death.
However, the CreER animals were left unaffected when tamoxifen was added later in life. Showing that Cre is damaging to neural progenitor cells before birth and that some types of hydrocephalus are due to defects in neural precursor proliferation.

58
Q

What issues did Hofman et al. run into in 2006 regarding transgenic animals

A

1/3 of those pigs studied with a lentiviral injection of transgenes experienced epigenetic modifications and were methylated to silence them.

59
Q

What does Simmons 2013 tell us about hybrid dysgenesis

A

P elements are transposable elements meaning whole sections of DNA can be moved elsewhere autonomously.
The P element codes its own transposase to allow it to be cleaved and repositioned elsewhere. They are found in humans and can be used for enhancer trapping - inserts P element near enhancer to study activity of the enhancer and the genes associated with it

60
Q

What does Nicholas et al state regarding HIV thymidine kinase in 2003

A

The herpesvirus thymidine kinase gene has also been used as a “suicide gene” as a safety system in gene therapy experiments, allowing cells expressing the gene to be killed using ganciclovir. This is desirable in case the recombinant gene causes a mutation leading to uncontrolled cell growth (insertional mutagenesis).

Has been used in animals to kill of tumour cells

61
Q

How can inverted loxP sites be used in condition cell ablation

Extra reading - Kmita and Gregoire 2008

A

Previously shown that loss of the loxP carrying chromosome can occur when loxP sites are arranged in inverse orientation. By adding cre-mediated recombination it not only causes chromosomal loss but also triggers apoptosis.

Targeted recombination between inverted loxP (TRIP) triggers cell death in proliferating cre-expressing cells and can therefore be used to ablate specific tissue types where cre is expressed

62
Q

Give an example where inverted loxP sites are being used to show cell apoptosis

A

Crossed mice having a set of loxP sites in inverted orientation
within the HoxD gene cluster with mice carrying a Cre transgene expressed primarily in developing limbs We found that limb buds of invloxP/; Prx1-Cre embryos were severely reduced in size as compared with wild-type ones.
In contrast, embryos carrying only the invloxP allele OR the Prx1-Cre
transgene were indistinguishable from wild-type embryos and were used as controls in subsequent analyses.

13,000 cre-positve GFP-positive cells remained per mutant limb bud,
whereas control buds contained 470,000 GFP-positive cells
This suggests cell death occurred as a result of chromosome elimination

63
Q

What is the role of exon5a in PAX6 gene
WIDER READING - Signh et al 2002

A

Found out role of exon 5a was by knocking only this section out of Pax6 gene - needed to make iris.

64
Q

How do we target cre-recombinase to the tissue of interest?

A

Drive Cre from the promoter of a gene that

Is only expressed in the tissue you want (quite rare) OR

Is expressed in areas that overlap with your tissue of interest

OR

Create a ‘Designer’ Promoter…

Make Transgenic Mouse carrying Cre on your Promoter, and Check Expression

65
Q

(If you are making a Cre trans gene, by the injection of DNA into a nucleus of a cell, what is the rate limiting step?) already covered more fancy words

A

the isolation and cloning of a reliable and robust promoter construct that drives strong Cre expression only in the places where you want it, has been the rate-limiting step for production of tissue-specific knockouts

66
Q

What is tamoxifen induction of cre expression?

A

fusion protein combining activity of cre and a mutant form of ligand binding domain of oestrogen receptor

67
Q

does cre-ER bind oestrogen?

A

no

68
Q

what does cre-ER bind?

A

4-OH-tamoxifen

69
Q

what happens to cre-ER if no tamoxifen?

A

Hsp90 grabs cre-ER and keeps it in cytoplasm so no recombination in nucleus

70
Q

what happens to cre-Er if tamoxifen present?

A

cre-TRM released from Hsp90 and recombination

Intercal - Advanced molecules, membranes and cells (31 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions