Medical and agricultural applications of gene technology Flashcards
Describe transient transformation
- 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
Describe stable transformation
- 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
Describe CaPO4 precipitation
- 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
What are the advantages of CaPO4 precipitation
- quick, cheap and simple
- not vector dependent
- can assay transient expression or score for stable integration
What does scoring for stable integration require?
a selectable marker
What are the limitations of CaPO4 precipitation?
essentially only used for mammalian cell lines
Describe electroporation
- 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
Describe the electric field of electroporation
- 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
What are the advantages of electroporation?
- 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
Give an example of electroporation use
in ESCs when making knockout/in mice
How is integration targeted when making knockout/in mice
homologous recombination
What are the limitations of electroporation?
- expensive equipment
- can only be used for single cells
- must be able to regenerate from a single cell is studying multicellular organisms
Describe making knockout/in mice
- transformed stem cells are injected into blastocyst
- implanted into surrogate mother
Describe microinjection - the basics
- used to transform various organisms including
nematodes, insects, fish, amphibians and mammals - frequently used to make transgenic mice
Describe microinjection - the specifics
- 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
What are the advantages of microinjection?
- not vector-dependent
- allows (usually random) stable integration
What are the limitations of microinjection?
- expensive (equipment and animal husbandry)
- requires skill
- labour intensive
Describe microprojectile bombardment
- particularly useful for plant transformation
- tungsten or gold particles coated with DNA fired into tissue using compressed gas as the propellant, under vacuum
Why is micro projectile bombardment useful for plant transformation
plant cells have tough cell wall
Describe the gene gun
initially used as an air rifle
Describe the stable transformation of maize using a gene gun
- 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
Describe the advantages of microprojectile bombardment
- 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)
Describe the limitations of micro projectile bombardment
- expensive equipment
- best suited for use with robust cells (such as plants)
Describe viral vectors for mammalian expression
- 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)
Describe the adenovirus for mammalian expression
- 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
Describe the lentiviruses for mammalian expression
- 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)
lentiviruses
subgroup of retroviruses
Give the advantages of viral vectors for mammalian expression
- 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
Give the limitations of viral vectors for mammalian expression
- vector-dependent
- potential activation of cellular oncogenes
- non-integrating vectors offer less stable expression, particularly in dividing cells
Describe Agrobacterium-mediated plant transformation
- 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
The most common method for transforming plants uses
the parasitic bacterium Agrobacterium tumefaciens
vir
virulence
T-DNA
- 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
Ti plasmid
Tumour inducing plasmid
A. tumefaciens causes
crown gall disease
Describe binary vector systems
separate the vir and T-DNA regions into two separate plasmids
Give the advantages of Agrobacterium-mediated plant transformation
- widely and routinely used - many vector options available
- enables stable integration
Give the limitations of Agrobacterium-mediated plant transformation
- 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
Describe the T-DNA vector
- 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
Describe the Ti plasmid
- ORI
- opine catabolism
- right border
- opine
- cytokinin
- plant hormones
- left border
- vir genes
When transferring DNA, one might wish to…
- 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
How can an endogenous gene be repressed by RNAi?
need cDNA sequence of endogenous gene
How can an endogenous gene be overexpressed by a constitutive promotor?
need cDNA sequence of endogenous gene
How can a novel gene be expressed by a constitutive promotor?
needs sequence of novel gene
How can a gene be expressed in a defined spatiotemporal context?
needs cDNA sequence of relevant gene and a suitable promotor
Describe choice of promotor for constitutive gene expression
- depends on system
- viral promoters and promoters of ubiquitously-expressed native genes are commonly used
List some commonly used promotors for mammalian cells
- SV40 virus
- cytomegalovirus
- ubiquitin
- actin
List some commonly used promotors for drosophila
- COPIA transposable element
- actin
List some commonly used promotors for yeast
- alcohol dehydrogenase
- cyclins
List some commonly used promotors for plants
- 35S cauliflower mosaic virus
- ubiquitin
- actin
Describe natural RNAi-mediated suppression of gene expression
- 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
What is the native function of RNAi
- antiviral defence mechanism in eukaryotes
- recognises dsRNA
siRNAs
- small interfering RNAs
- approximately 20nts
Describe artificial RNAi-mediated suppression of gene expression
- 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
RISC
RNA-Induced silencing complex
Describe RNAi constructs morphologically
- promotor
- target sequence
- intron spacer
- inverted repeat of target sequence corresponding to gene of interest
- terminator
Describe RNAi constructs functionally
- construct can be stably introduced into the genome of the organism of interest
- designed such that encoded RNA forms a hairpin loop containing dsRNA
Describe RNAi constructs physiologically
- 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
Describe genome editing using ‘designer nucleases’
- 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
Describe ZFNs
- zinc finger nucleases
- DNA-binding protein domain followed by nuclease
Describe TALENs
- Transcription Activator-Like Effector Nuclease
- nuclease followed by DNA-binding protein domain
Describe CRISPR-Cas9
RNA and Cas9 nuclease
Describe the different downstream effects of gene editing
- DNA ds break at nuclease target site
- homology-directed repair or non-homologous end-joining
Non-homologous end-joining results in
gene disruption
Homology-directed repair results in
- exogenous donor template
- gene correction (knock-in)
- transgene insertion (knock-in)
Describe the agricultural applications of RNAi
- polygalacturonase RNAi in tomatoes
Describe polygalactouronase
- 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
Describe glyphosate resistant crops
- crops transformed with a bacterial version of the enzyme
Describe glyphosate
- broad-spectrum herbicide
- targets EPSP synthase
Describe EPSP synthase
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)
Describe virus resistant papaya
- express PRSV coat protein gene
- interferes with viral replication due to gene silencing
- provides immunity against the virus
- has been successfully applied in Hawaii
Describe blight resistant potato
expresses two genes from a Mexican wild potato that give resistance to late blight
Describe ‘golden rice’
- enhanced nutrients
- engineered to express two genes from the beta-carotene (provitamin A) pathway in grain
Describe insect resistant cotton
- express Bt toxin
- controls only pests eating
the plants: discriminative
Describe ‘cross protection’
resistance is achieved by applying mild form of virus
Describe global vitamin A deficiency
- > 1 million children die due to vitamin A deficiency each year
- many more have sight loss
Describe Bt toxin
- derived from soil bacterium Bacillus thuringiensis
- affects insect digestive system
Describe the use of cell transformation in insulin production
- human insulin produced in bacteria
- for diabetes treatment
Describe the use of cell transformation in human growth hormone production
- in bacteria
- for treatment of pituitary dwarfism
Describe the use of cell transformation in Hepatitis B vaccine production
- in yeast
- vaccine contains recombinant coat protein only
Describe the use of cell transformation in Factor VIII and tPA production
made in yeast
tPA
- tissue plasminogen activator (tPA)
- declotting agent
- used to treat blood clots; pulmonary embolism or stroke
Factor VIII
- clotting agent
- used to treat haemophilia
Describe the production of protein in lactating mammals
- 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
Beta-lactoglobulin promotor
mammary-gland specific
What are some issues associated with gene therapy?
- somatic or germline
- targeting
- immune evasion
- expression regulation
Describe gene therapy for SCID
- 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
Describe SCID
- severe combined immunodeficiency
- compromised immune system due to absence of function T and B cells
- single-gene defects in X-linked IL2RG, or ADA
Describe gene therapy for cystic fibrosis
deliver aerosol non-mutated gene to lung epithelia using liposomes from a nebuliser, in vivo
Describe cystic fibrosis
- patients produce thick viscous mucous
- prone to lung infections
- defect in CFTR
CFTR
cystic fibrosis transmembrane conductance regulator ion channel gene
Describe gene therapy for beta-thalassemia or sickle cell disease
deliver non-mutated gene into haematopoietic stem cells from pateit ex vivo using a lentivirus vector
Describe beta-thalassemia and sickle cell disease
- pateints make less or defective haemoglobin or fewer or defective RBCs respectively
- causes anaemia
- results in defects in the beta-globing gene
Describe gene therapy for acute lymphoblastic leukaemia
- bespoke therapy
- off-the-shelf therapy
Describe bespoke therapy for acute lymphoblastic leukaemia
- patient’s own T cells engineered by lentivirus to target cancer / B cells
- very expensive
- not suitable for very ill patients
Describe acute lymphoblastic leukaemia
malignant B lymphoblasts
Describe ‘off-the-shelf’ therapy for acute lymphoblastic leukaemia
- 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)
Describe gene therapy for sickle cell disease
- CTX0001
- patient’s haemotopoietic stem cells modified ex vivo by CRISPR to inactivate the BCL11A gene
- increases HbF expression
