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Vectors Flashcards

1
Q

what is the genetic map importance

A

fundamental resource in the future of biomedical research into disease

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2
Q

how many base pairs does the human chromosome house

A

3 billion base pairs of DNA that contains only 30,000 - 40,000 protein-coding genes (5% of the genome)

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3
Q

what part of genetic disease is good for gene therapy

A

Most of the genetic diseases are defined by a mutation in one gene

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4
Q

what makes some diseases harder to cure

A

some diseases e.g. diabetes and asthma, more then one gene so understanding disease, is more difficult and hence so to a cure

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5
Q

what is sickle-cell anemia

A

inherited, chronic blood disease in which of red blood cells become crescent shaped and function abnormally

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6
Q

why do patients become anemic due to sickle-cell

A

Patients become anemic because of decreased ability of red blood cells to provide adequate oxygen supplies to body tissues, usually die between the ages of 20-40

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7
Q

what is the ratio of sickle cell anemia of african origin

A

1:500

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8
Q

what causes sickle cell-anemia

A

Autosomal recessive mutation in HBB (human beta-globin) gene, chromosome 11
normal haemoglobin is called A

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9
Q

what is the defect in sickle-cell

A

Haemoglobin molecule-S caused by a single base change results in a E –> V causes sickled red blood cells

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10
Q

what is A/S sickle cell

A

heterozygotes, have sickle cell trait are carriers, (have mild anemia, intermediate inheritance)

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11
Q

what is S/S sickle cell

A

S/S are homozygotes, have disease

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12
Q

what is the treatment for sickle cell

A

no cure for sickle cell, since the gene has been mapped, a prenatal diagnosis is available using RFLP analysis

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13
Q

what does RFLP analysis allow

A

This allows parents to determine if fetus will be A/A, A/S or S/S
whether to keep or abort

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14
Q

what is A/A sickle cell

A

normal adult haemoglobin

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15
Q

why is sickle cell still present

A

Sickle cell people are resistant to malaria so provides an advantage

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16
Q

what is gene therapy

A

transfer of selected genes into a host with the hope of curing a disease state

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17
Q

what is traditional pharmaceutical development

A

Pharamceuticals that will interact with gene products

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18
Q

what is gene therapy

A

if gene is corrected

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19
Q

what is cystic fibrosis treated with

A

antibiotics

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20
Q

what does antibiotic treatment of cystic fibrosis do

A

clear lungs - traditional

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21
Q

what is the gene therapy of cystic fibrosis

A

genetic treatment correct DNAse gene

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22
Q

what is SCID

A

Severe combined Immunodificiency Disease

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23
Q

what has caused SCID

A

number of mutations have been attributed to causing SCID

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24
Q

what causes SCID

A

Genetic mutations occurs on Autosomes 19 and 20 and also on X Chromosome (where boys would be effected, since girls have 2 copies of the X chromosome, second of which is likely to be normal)

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25
what are SCID children called
bubble babies
26
why are SCID children called bubble babies
they must live in a sterile environment as they can not cope with infections
27
where are viral vectors used
Used in both ex vivo or in vivo therapies
28
how do viral vectors propagate
Normally are replication defective, need to propagate in special cell lines
29
what do vectors include
Retroviral adenoviral Adeno-associated Herpes virus
30
what is ex vivo gene transfer
transfer of genetic material to cells isolated from host following transfer cells are implanted back indirect gene transfer
31
what is the advantage of ex vivo gene transfer
can take out cancer cells
32
what is in-vivo gene transfer
Transfer of genetic material to cells located within host | aka direct gene transfer
33
what are the advantages of retroviral vectors
- Easy to work with, plasmids are easy to engineer - Packaging lines available to simplify generation of viral particles - Gives high number of transfected cells
34
what are the disadvantages of retroviral vectors
- Limited gene capacity (6KB) | - Integrate into human genome and therefore are potentially oncogenic (cancer) due to insertional mutagenisis
35
what happens in ex-vivo gene therapy
1. Bone marrow cells removed 2. Bone marrow enriched to isolate parent cells or immune system 3. Cells infected with virus containing gene therapy 4. Cells take up gene, normal version of which is missing in boys 5. Cells returned to baby boy 6. After 2 weeks immune system develops top protect baby from infection ADA expressing cells are transferred back
36
what is one of the mutations in SCID
in gene Adenosine deaminase
37
what is ADA
adenosine deaminase
38
what effect does a loss of function mutation in ADA have
causes a build up of adenosine which causes death of B and T cells lymphocytes which are crucial to our ability to fight infections
39
what are viral vectors grown with
viral vectors have been grown which contain ADA
40
where are viral vectors that contain ADA used
transferred to totipotent stem cells (bone marrow cells) | cells were the transferred to patient and ADA expressing cells corrects defect
41
what happens in retrovirus life cycle
Virus comes in, inserts ssRNA in which is converted to DNA integrated into genome = provirus As a provirus it can express its genes Normally in retrovirus late phase it transcribes, translates is assembled to be moved out of cell
42
how are the early and late phases differentiated
provirus stays in cell in early phase and passes out in late phase
43
what would happen in gene therapy retrovirus cycle
In gene therapy would not want it to leave and make a virus
44
what happens in early retrovirus life cycle
Produce DNA copy of RNA genome by using RT carried within virion transport of vDNA to nucleus integrating vDNA into host genome
45
what happens in late retrovirus life cycle
viral mRNA transcription and progeny viral RNA from vDNA, driven by promoter in 5’ Long Terminal Repeat Translation in cytoplasm of Viral proteins Formation and packaging of viral capsid with two RNA strands and RT molecules Budding of enveloped virons to exterior of cell
46
what is gag region
gag region encodes capsid proteins
47
what is pol region
pol encodes reverse transcriptase and integrase proteins
48
what is the env region
env encodes envelope and proteins needed for receptor recognition and envelope anchoring
49
what is psi
packaging signal
50
what is the long terminal region important for
LTR is important in initiating viral DNA synthesis and integration
51
what happens when virus infected into human cells
Once infected into human cells virus will integrate but can't make virus as doesn't contain genes for virus (gag, pol and env)
52
what happens in MMLV ADA expressing virus
package vector RNA (encoding ADA) into viruses which then can then be used to deliver genes to human cells
53
what is used to make a 'hollowed out' virus genome
MMLV vector
54
what does MMLV vector genome contain
packaging signals psi Ψ+ and LTRs from Virus
55
what selectable marker gene does MMLV vector contain
neomysin
56
what is the transgene in MMLV vector
adenosine deaminase
57
why can MMLV vector get out of the cell
Does not encode viral genes gag, pol and env
58
what is a selectable marker
Often to select transgene, one must co-express a selectable marker, allowing only cells expressing transgene to be expressed
59
what is a transgene
selected gene tested in a gene transfer experiment
60
what does a viral vector contain
Psi packaging signal Long terminal repeats Gene x – e.g. ADA Promoter e.g. SV40 Important for transcription enhancement and drives neomycin resistance
61
what happens in MMLV therapy for SCID
1. MMLV vector expressing ADA is constructed 2. Vector transfected onto packaging cell line, producing a Producer cell line 3. Virus produced from Producer cell line that expresses ADA 4. virus used to infect stem cells, isolated from bone marrow 5. cells, expressing ADA are then transferred to SCID patient
62
what does the packaging line encode
gag pol env
63
what does the packaging line synthesise
viral proteins but since lines contain no encapsidation signal (ΔΨ) in either gene only empty capsids are produced
64
what is the producer cell line in MMLV
producing recombinant virus which contains ADA and neomycin resistance
65
how is a retroviral vector RNA made
packaging cell line, want to add: ADA, long terminal repeats and neomycin Genes encapsulated into a virus by psi vector transfected onto packaging cell producing specific producer cell line virus is produced encodes ADA
66
what happens after transfection of packaging cell line with ADA retroviral vector DNA
DNA integrates into chromosome
67
what is transcribed and put into viral capsids
Full length RNA from the retrovirus vector that carries the encapsidation signal psi (Ψ+)
68
can viral particles replicate after made
can only replicate on packaging cell lines as don’t have gag, pol and env genes to make a virus
69
what is the ADA expressing virus used for
infect stem cells isolated from bone marrow cells
70
once virus in stem cell can ADA and neo still be expressed
Once in stem cell cant make anymore virus can just express these genes ADA expressing virus can integrate and express (ADA and neo) can not make a virus can only do so in packaging cell line, stem cells do not express gag, pol and env

Applied Genetics (20 decks)

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