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Applied Genetics > Vectors > Flashcards

Vectors Flashcards

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Biology 101 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
Q

what are SCID children called

A

bubble babies

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26
Q

why are SCID children called bubble babies

A

they must live in a sterile environment as they can not cope with infections

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27
Q

where are viral vectors used

A

Used in both ex vivo or in vivo therapies

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28
Q

how do viral vectors propagate

A

Normally are replication defective, need to propagate in special cell lines

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29
Q

what do vectors include

A

Retroviral
adenoviral
Adeno-associated
Herpes virus

30
Q

what is ex vivo gene transfer

A

transfer of genetic material to cells isolated from host
following transfer cells are implanted back
indirect gene transfer

31
Q

what is the advantage of ex vivo gene transfer

A

can take out cancer cells

32
Q

what is in-vivo gene transfer

A

Transfer of genetic material to cells located within host

aka direct gene transfer

33
Q

what are the advantages of retroviral vectors

A
  • 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
Q

what are the disadvantages of retroviral vectors

A
  • Limited gene capacity (6KB)

- Integrate into human genome and therefore are potentially oncogenic (cancer) due to insertional mutagenisis

35
Q

what happens in ex-vivo gene therapy

A
  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
Q

what is one of the mutations in SCID

A

in gene Adenosine deaminase

37
Q

what is ADA

A

adenosine deaminase

38
Q

what effect does a loss of function mutation in ADA have

A

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
Q

what are viral vectors grown with

A

viral vectors have been grown which contain ADA

40
Q

where are viral vectors that contain ADA used

A

transferred to totipotent stem cells (bone marrow cells)

cells were the transferred to patient and ADA expressing cells corrects defect

41
Q

what happens in retrovirus life cycle

A

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
Q

how are the early and late phases differentiated

A

provirus stays in cell in early phase and passes out in late phase

43
Q

what would happen in gene therapy retrovirus cycle

A

In gene therapy would not want it to leave and make a virus

44
Q

what happens in early retrovirus life cycle

A

Produce DNA copy of RNA genome by using RT carried within virion
transport of vDNA to nucleus
integrating vDNA into host genome

45
Q

what happens in late retrovirus life cycle

A

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
Q

what is gag region

A

gag region encodes capsid proteins

47
Q

what is pol region

A

pol encodes reverse transcriptase and integrase proteins

48
Q

what is the env region

A

env encodes envelope and proteins needed for receptor recognition and envelope anchoring

49
Q

what is psi

A

packaging signal

50
Q

what is the long terminal region important for

A

LTR is important in initiating viral DNA synthesis and integration

51
Q

what happens when virus infected into human cells

A

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
Q

what happens in MMLV ADA expressing virus

A

package vector RNA (encoding ADA) into viruses which then can then be used to deliver genes to human cells

53
Q

what is used to make a ‘hollowed out’ virus genome

A

MMLV vector

54
Q

what does MMLV vector genome contain

A

packaging signals psi Ψ+ and LTRs from Virus

55
Q

what selectable marker gene does MMLV vector contain

A

neomysin

56
Q

what is the transgene in MMLV vector

A

adenosine deaminase

57
Q

why can MMLV vector get out of the cell

A

Does not encode viral genes gag, pol and env

58
Q

what is a selectable marker

A

Often to select transgene, one must co-express a selectable marker, allowing only cells expressing transgene to be expressed

59
Q

what is a transgene

A

selected gene tested in a gene transfer experiment

60
Q

what does a viral vector contain

A

Psi packaging signal
Long terminal repeats
Gene x – e.g. ADA
Promoter e.g. SV40 Important for transcription enhancement and drives neomycin resistance

61
Q

what happens in MMLV therapy for SCID

A
  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
Q

what does the packaging line encode

A

gag
pol
env

63
Q

what does the packaging line synthesise

A

viral proteins but since lines contain no encapsidation signal (ΔΨ) in either gene only empty capsids are produced

64
Q

what is the producer cell line in MMLV

A

producing recombinant virus which contains ADA and neomycin resistance

65
Q

how is a retroviral vector RNA made

A

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
Q

what happens after transfection of packaging cell line with ADA retroviral vector DNA

A

DNA integrates into chromosome

67
Q

what is transcribed and put into viral capsids

A

Full length RNA from the retrovirus vector that carries the encapsidation signal psi (Ψ+)

68
Q

can viral particles replicate after made

A

can only replicate on packaging cell lines as don’t have gag, pol and env genes to make a virus

69
Q

what is the ADA expressing virus used for

A

infect stem cells isolated from bone marrow cells

70
Q

once virus in stem cell can ADA and neo still be expressed

A

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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