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Hilary Term 1 > Medical and agricultural applications of gene technology > Flashcards

Medical and agricultural applications of gene technology Flashcards

1
Q

Describe transient transformation

A
  • introduced DNA is not integrated into host genome
  • diluted with every host cell cycle
  • suitable for short-term expression or use with non-rapidly dividing cells
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2
Q

Describe stable transformation

A
  • introduced DNA is integrated into the host genome
  • is replicated as the cells divide
  • often non-targeted
  • targeted integration possible in some species via homologous recombination
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3
Q

Describe CaPO4 precipitation

A
  • plasmid DNA put in phosphate buffer
  • add CaCL2
  • DNA coprecipitates alongside CaPO4
  • add DNA to mammalian culture cells
  • precipitate binds to cell surface
  • is endocytosed
  • incubate for 4-16hrs at 37 degrees
  • add fresh medium to cells
  • remove DNA solution
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4
Q

What are the advantages of CaPO4 precipitation

A
  • quick, cheap and simple
  • not vector dependent
  • can assay transient expression or score for stable integration
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5
Q

What does scoring for stable integration require?

A

a selectable marker

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6
Q

What are the limitations of CaPO4 precipitation?

A

essentially only used for mammalian cell lines

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7
Q

Describe electroporation

A
  • plasmid placed in electroporation cuvette containing cations and anions, and put under an electric filed
  • pores form in the membrane, allowing DNA to enter
  • membrane heals
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8
Q

Describe the electric field of electroporation

A
  • very short, intense pulse
  • 2500V/cm for just a few milliseconds for bacteria
  • lower for animal and plant cells
  • causes polarisation of the cell membrane
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9
Q

What are the advantages of electroporation?

A
  • quick
  • not vector dependent
  • used for bacteria, yeast, plant protoplasts and mammalian cells
  • can assay transient expression or score for stable integration, depending on system
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10
Q

Give an example of electroporation use

A

in ESCs when making knockout/in mice

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11
Q

How is integration targeted when making knockout/in mice

A

homologous recombination

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12
Q

What are the limitations of electroporation?

A
  • expensive equipment
  • can only be used for single cells
  • must be able to regenerate from a single cell is studying multicellular organisms
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13
Q

Describe making knockout/in mice

A
  • transformed stem cells are injected into blastocyst
  • implanted into surrogate mother
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14
Q

Describe microinjection - the basics

A
  • used to transform various organisms including
    nematodes, insects, fish, amphibians and mammals
  • frequently used to make transgenic mice
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15
Q

Describe microinjection - the specifics

A
  • transgene injected into fertile male pronucleus
  • transgene integrates randomly into genome
  • one-cell embryos collected
  • embryos injected into sterile pseudopregnant female
  • live birth test for transgene
  • creates transgenic founder animal
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16
Q

What are the advantages of microinjection?

A
  • not vector-dependent
  • allows (usually random) stable integration
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17
Q

What are the limitations of microinjection?

A
  • expensive (equipment and animal husbandry)
  • requires skill
  • labour intensive
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18
Q

Describe microprojectile bombardment

A
  • particularly useful for plant transformation
  • tungsten or gold particles coated with DNA fired into tissue using compressed gas as the propellant, under vacuum
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19
Q

Why is micro projectile bombardment useful for plant transformation

A

plant cells have tough cell wall

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20
Q

Describe the gene gun

A

initially used as an air rifle

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21
Q

Describe the stable transformation of maize using a gene gun

A
  • scutellum in an embryonic leaf in the seed isolated at day 0
  • particle bombardment at day 4
  • selection of transformants (relies on antibiotic-resistance markers in the introduced DNA) in 3 week bursts
  • transfer to soil after 12 weeks
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22
Q

Describe the advantages of microprojectile bombardment

A
  • quick
  • can be used on any tissue
  • not vector-dependent
  • can assay transient expression or score for integration
  • can deliver DNA to organelles (such as chloroplasts)
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23
Q

Describe the limitations of micro projectile bombardment

A
  • expensive equipment
  • best suited for use with robust cells (such as plants)
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24
Q

Describe viral vectors for mammalian expression

A
  • various mammalian viruses can be adapted for use as vectors
  • replication-defective (genes responsible for replication removed)
  • used to deliver DNA to cells or intact organisms (e.g., in gene therapy)
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25
Q

Describe the adenovirus for mammalian expression

A
  • dsDNA virus
  • does not integrate into host genome
  • allows short/medium-term expression (weeks/months)
  • can deliver strong transgene expression in non- or slowly-dividing cells
  • high transgene capacity (>30kb)
  • gets lost more quickly in rapidly-dividing cells
  • can provoke brisk immune response
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26
Q

Describe the lentiviruses for mammalian expression

A
  • ssRNA virus
  • integrates into host genome
  • allows long-term expression (years)
  • can integrate into genome of non-dividing cells, unlike other retroviruses (which require breakdown of the nuclear envelope)
  • unpredictable site of integration (potential oncogene activation)
  • minimal immune response
  • limited transgene capacity (approximately 8kb)
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27
Q

lentiviruses

A

subgroup of retroviruses

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28
Q

Give the advantages of viral vectors for mammalian expression

A
  • effective delivery of DNA to cells, in vivo or ex vivo
  • applicable to many systems (viruses and vector derivatives are v diverse)
  • high levels of transgene expression
  • long-term, stable integration
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29
Q

Give the limitations of viral vectors for mammalian expression

A
  • vector-dependent
  • potential activation of cellular oncogenes
  • non-integrating vectors offer less stable expression, particularly in dividing cells
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30
Q

Describe Agrobacterium-mediated plant transformation

A
  • wounded plant cells release factors that stimulate transcription of vir genes on the bacterial Ti plasmid
  • enables bacteria to infect plant tissue, and transfer T-DNA region of Ti into the plant genome
  • T-DNA can be disarmed and adapted for use as a vector
  • transfers gene of interest into plant genome
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31
Q

The most common method for transforming plants uses

A

the parasitic bacterium Agrobacterium tumefaciens

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32
Q

vir

A

virulence

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33
Q

T-DNA

A
  • Transfer-DNA
  • codes genes for hormones to promote cell proliferation (gall formation), and opines which nourish the bacteria
  • hormone and opine genes can be replaced with selectable marker and gene of interest
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34
Q

Ti plasmid

A

Tumour inducing plasmid

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35
Q

A. tumefaciens causes

A

crown gall disease

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36
Q

Describe binary vector systems

A

separate the vir and T-DNA regions into two separate plasmids

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37
Q

Give the advantages of Agrobacterium-mediated plant transformation

A
  • widely and routinely used - many vector options available
  • enables stable integration
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38
Q

Give the limitations of Agrobacterium-mediated plant transformation

A
  • limited host range (extensive in dicots, but many monocots and gymnosperms not readily infected)
  • can be time consuming, depending on species
  • site of genome integration is random
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39
Q

Describe the T-DNA vector

A
  • origin of replication
  • right T-DNA border
  • terminator
  • gene of interest
  • promotor
  • plant selectable marker gene
  • terminator
  • left T-DNA border
  • bacterial susceptible marker gene
  • virulence region
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40
Q

Describe the Ti plasmid

A
  • ORI
  • opine catabolism
  • right border
  • opine
  • cytokinin
  • plant hormones
  • left border
  • vir genes
41
Q

When transferring DNA, one might wish to…

A
  • repress endogenous gene expression by RNAi
  • overexpress an endogenous gene with a constitutive promotor
  • express a novel gene with a constitutive promotor
  • express a gene in a defined spatiotemporal context
42
Q

How can an endogenous gene be repressed by RNAi?

A

need cDNA sequence of endogenous gene

43
Q

How can an endogenous gene be overexpressed by a constitutive promotor?

A

need cDNA sequence of endogenous gene

44
Q

How can a novel gene be expressed by a constitutive promotor?

A

needs sequence of novel gene

45
Q

How can a gene be expressed in a defined spatiotemporal context?

A

needs cDNA sequence of relevant gene and a suitable promotor

46
Q

Describe choice of promotor for constitutive gene expression

A
  • depends on system
  • viral promoters and promoters of ubiquitously-expressed native genes are commonly used
47
Q

List some commonly used promotors for mammalian cells

A
  • SV40 virus
  • cytomegalovirus
  • ubiquitin
  • actin
48
Q

List some commonly used promotors for drosophila

A
  • COPIA transposable element
  • actin
49
Q

List some commonly used promotors for yeast

A
  • alcohol dehydrogenase
  • cyclins
50
Q

List some commonly used promotors for plants

A
  • 35S cauliflower mosaic virus
  • ubiquitin
  • actin
51
Q

Describe natural RNAi-mediated suppression of gene expression

A
  • Dicer cleaves dsRNA into siRNAs
  • bound by the RISC complex
  • target RISC to the complementary target mRNA
  • RISC leaves mRNA
  • protein not produced even when target gene is actively transcribed
52
Q

What is the native function of RNAi

A
  • antiviral defence mechanism in eukaryotes
  • recognises dsRNA
53
Q

siRNAs

A
  • small interfering RNAs
  • approximately 20nts
54
Q

Describe artificial RNAi-mediated suppression of gene expression

A
  • experimentally delivered dsRNA
  • Dicer coupling
  • cleavage of dsRNA into siRNAs
  • uncoupling of Dicer
  • unwinding
  • recognition of target mRNA
  • coupling of RISC
  • cleavage of target mRNA
  • uncoupling of RISC
  • fragments of target mRNA
55
Q

RISC

A

RNA-Induced silencing complex

56
Q

Describe RNAi constructs morphologically

A
  • promotor
  • target sequence
  • intron spacer
  • inverted repeat of target sequence corresponding to gene of interest
  • terminator
57
Q

Describe RNAi constructs functionally

A
  • construct can be stably introduced into the genome of the organism of interest
  • designed such that encoded RNA forms a hairpin loop containing dsRNA
58
Q

Describe RNAi constructs physiologically

A
  • transcription and annealing to form hairpin RNA
  • intron spliced out
  • target dsRNA produced
  • dsRNA processed by Dicer, leading to RNAi-mediated silencing of the target gene
59
Q

Describe genome editing using ‘designer nucleases’

A
  • editing of a gene of interest in situ
  • sequence-specific guiding mechanism to target a nuclease to a gene of interest
  • guide can be protein or RNA; can be engineered to achieved desired specificity
  • once in situ, nuclease creates a ds break within the target gene
  • many different outcomes
60
Q

Describe ZFNs

A
  • zinc finger nucleases
  • DNA-binding protein domain followed by nuclease
61
Q

Describe TALENs

A
  • Transcription Activator-Like Effector Nuclease
  • nuclease followed by DNA-binding protein domain
62
Q

Describe CRISPR-Cas9

A

RNA and Cas9 nuclease

63
Q

Describe the different downstream effects of gene editing

A
  • DNA ds break at nuclease target site
  • homology-directed repair or non-homologous end-joining
64
Q

Non-homologous end-joining results in

A

gene disruption

65
Q

Homology-directed repair results in

A
  • exogenous donor template
  • gene correction (knock-in)
  • transgene insertion (knock-in)
66
Q

Describe the agricultural applications of RNAi

A
  • polygalacturonase RNAi in tomatoes
67
Q

Describe polygalactouronase

A
  • enzyme that degrades pectin (component of the plant cell wall)
  • causes tomato fruit softening
  • fruit harvested prematurely
  • fruitless flavourful as those harvested on the vine
68
Q

Describe glyphosate resistant crops

A
  • crops transformed with a bacterial version of the enzyme
69
Q

Describe glyphosate

A
  • broad-spectrum herbicide
  • targets EPSP synthase
70
Q

Describe EPSP synthase

A

converts shikimic acid-3-phosphate to 5-enolpyruvyl shikmic acid-3-phosphate, which converts to chorismic acid (latterly phenylalanine) and anthranilic acid (latterly tyrosine and tryptophan)

71
Q

Describe virus resistant papaya

A
  • express PRSV coat protein gene
  • interferes with viral replication due to gene silencing
  • provides immunity against the virus
  • has been successfully applied in Hawaii
72
Q

Describe blight resistant potato

A

expresses two genes from a Mexican wild potato that give resistance to late blight

73
Q

Describe ‘golden rice’

A
  • enhanced nutrients
  • engineered to express two genes from the beta-carotene (provitamin A) pathway in grain
74
Q

Describe insect resistant cotton

A
  • express Bt toxin
  • controls only pests eating
    the plants: discriminative
75
Q

Describe ‘cross protection’

A

resistance is achieved by applying mild form of virus

76
Q

Describe global vitamin A deficiency

A
  • > 1 million children die due to vitamin A deficiency each year
  • many more have sight loss
77
Q

Describe Bt toxin

A
  • derived from soil bacterium Bacillus thuringiensis
  • affects insect digestive system
78
Q

Describe the use of cell transformation in insulin production

A
  • human insulin produced in bacteria
  • for diabetes treatment
79
Q

Describe the use of cell transformation in human growth hormone production

A
  • in bacteria
  • for treatment of pituitary dwarfism
80
Q

Describe the use of cell transformation in Hepatitis B vaccine production

A
  • in yeast
  • vaccine contains recombinant coat protein only
81
Q

Describe the use of cell transformation in Factor VIII and tPA production

A

made in yeast

82
Q

tPA

A
  • tissue plasminogen activator (tPA)
  • declotting agent
  • used to treat blood clots; pulmonary embolism or stroke
83
Q

Factor VIII

A
  • clotting agent
  • used to treat haemophilia
84
Q

Describe the production of protein in lactating mammals

A
  • gene follows beta-lactoglobulin promotor
  • DNA injected into pronucleus of sheep ovum using holding pipette
  • implanted into foster mother
  • transgenic progeny identified by PCR
  • expression restricted to mammary tissue
  • product secreted into milk
  • fractionate milk products
85
Q

Beta-lactoglobulin promotor

A

mammary-gland specific

86
Q

What are some issues associated with gene therapy?

A
  • somatic or germline
  • targeting
  • immune evasion
  • expression regulation
87
Q

Describe gene therapy for SCID

A
  • deliver non-mutated gene into patient’s T- or haematopoietic stem cells ex vivo using a gamma retroviral vector
  • risks with oncogene expression leading to leukaemia
88
Q

Describe SCID

A
  • severe combined immunodeficiency
  • compromised immune system due to absence of function T and B cells
  • single-gene defects in X-linked IL2RG, or ADA
89
Q

Describe gene therapy for cystic fibrosis

A

deliver aerosol non-mutated gene to lung epithelia using liposomes from a nebuliser, in vivo

90
Q

Describe cystic fibrosis

A
  • patients produce thick viscous mucous
  • prone to lung infections
  • defect in CFTR
91
Q

CFTR

A

cystic fibrosis transmembrane conductance regulator ion channel gene

92
Q

Describe gene therapy for beta-thalassemia or sickle cell disease

A

deliver non-mutated gene into haematopoietic stem cells from pateit ex vivo using a lentivirus vector

93
Q

Describe beta-thalassemia and sickle cell disease

A
  • pateints make less or defective haemoglobin or fewer or defective RBCs respectively
  • causes anaemia
  • results in defects in the beta-globing gene
94
Q

Describe gene therapy for acute lymphoblastic leukaemia

A
  • bespoke therapy
  • off-the-shelf therapy
95
Q

Describe bespoke therapy for acute lymphoblastic leukaemia

A
  • patient’s own T cells engineered by lentivirus to target cancer / B cells
  • very expensive
  • not suitable for very ill patients
96
Q

Describe acute lymphoblastic leukaemia

A

malignant B lymphoblasts

97
Q

Describe ‘off-the-shelf’ therapy for acute lymphoblastic leukaemia

A
  • T cells from another person engineered by lentivirus to target cancer / B cells
  • engineered by TALEN to evade patient immune system (with a delta-T cell receptor) and an anti-rejection drug (deltaCD52)
98
Q

Describe gene therapy for sickle cell disease

A
  • CTX0001
  • patient’s haemotopoietic stem cells modified ex vivo by CRISPR to inactivate the BCL11A gene
  • increases HbF expression

Hilary Term 1 (61 decks)

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