Vitamin A and Rice initial + more recent transgenic mods
- Rice = major food source but Vit A deficient
- make golden rice
- introduce daffodil Phytoene synthase + lycopene beta cyclase
- introduce phytoene saturase (Erwinia)
- fuse all 3 genes with transit peptide –> chl delivery for mass expression –> allow rice to produce significant amount of beta Carotene
- beta carotene ingested –> consumer converts to vit A
newer strains of golden rice use phytoene synthase from corn –> high activity therefore more beta carotene
Vit B9 (folate) deficiency in rice
- limited wt production due to rate limiting steps
- insert arabidopsis B9 biosynthesis –> 100x increase to B9 folate
- use many transit peptides since enzymes have to chl, cytosol, mitochondria
tomato ripening - what genes, what do they do, how was flavrsavr tomato modified?
pTom5 = lycopene (causes ripening from green to red)
pTom6 = CW degradation (softening, sweetening)
pTom13 = ethylene (trigger ripening process)
excess ptom 13 ethylene = overripening
RNAi of ptom13 to downregulate ethylene accumulation. Normal ripening process, but delays threshold accumulation to begin rotting –> longer shelf life
Potatoes - transgenic starch reduction?
amylose + starch branching enzymes (SBE) = amylopectin
reduce amylopectin presence using RNAi of SBEs –> potatoe cells less dmaaged after sustained freeze/thaw cycles
transgenic trees for pulp production
- decrease lignin presence to make pulping easier
- RNAi knockdown of lignin = 45% less lignin + 15% more cellulose (compensate for less lignin)
- antisensed to 4-coumarate CoA ligase
trees became thicker/larger to compensate for lack of lignin
Why use plants to produce things?
- they produce products with the adequate post-trans mods –> not perfect, but very close (Plants > bacteria)
- cheaper to maintain than animal cells
- easy to scale up
- HIGH YIELD
transgenic plants - viral vectors - examples + how the virusses were modded
tobacco mosiac virus (TMV)
potato virus X (PVX)
disable coat proteins –> prevent virion synthesis therefore prevent lysis/harm
Retain viral replicase + viral expression pathways –> strong expression
agrobacterium transfection
Transgenic plants - Why not integrate the GOI into chromosomes?
- want to insert many copies of GOI –> integration may cause interference of chromosomal genes + risk of off targetting on chromosome
- chl DNA is maternally derived therefore no pollen HGT
If expressing the genes:
- chl mimics bacterial PTMs –> more likely to get fxning bacterial transgenic protein
- lots of chl = lots of constituitive GOI expression
plantibodies
plant synthesized antibodies
transgenic plants modified to carry human Ig heavy/light chains on viral vector –> high expression
fusion with excretion domain for ease of harvesting
edible vaccines - example + method
- modify crops/food to carry the vaccine peptides/proteins –> diffusion/uptake through GI into blood
- Eg cholera toxin vaccine using potatoes
- cholera toxin = B subuit (cell binding) + A2 linker peptide + A1 toxin peptide
- delivert subunit B to potatoes using agrobacterium –> vaccination
modifying cholera toxin for wider vaccinations
fuse rotoviral peptides to cholera toxin B subunit
replace A1 toxin with bacterial colony growth factor
therefore the edible vaccine allows immunization of cholera (B subunit), rotovirus and bacterial infection resistance
Ecoli modified to use HDR - What plasmids involved?
pCas9 plasmid - Cas9 + lambda red + kanR marker + temperature sensitive Ori
lambda-red confers HDR mechanisms
T sensitive Ori –> after genes are recombined into genome –> need to remove the construct
(retaining the pCas9 construct after recombination causes issues)
pTargetF = gRNA carrier (use for mutagenesis)
pTargetT = gRNA + ssDNA carrier (use for guided mutagenesis/insertions)
Ecoli modified to use HDR - confirmational testing - the premise
- transform a chloramphenicol resistant strain –> mutated with a gene insert to interrupt it
- Use pCas9 w/Lmabda red + cas9 + KanR
- use pTargetT w/gRNA + ssDNA encoding chloramphenicolR –> restore gene
mix and match diff combinations –> assess degree of chlorR
Ecoli modified to use HDR - confirmational testing - the results/interpretations on mutation efficiency
no cas9 = minimal resistance –> recombination still allowed small degree of mutation efficiency
no gRNA = small resistance –> due to random Cas9 randomly binding and initiating the cuts
no lambda red = 0 resistance –> no ability to recombine and restore fxn
no ssDNA chloramphenicol = 0 resistance –> no template to restore fxn
all elements present = lots of resistance = lots of mutation efficiency
Plant crispr - how and why? What molecule(s) used to specifically faciilitate this transfection?
agrobacterium transfection
delivery into chl w/o integration into chromosome (DNA free editting)
use preassembled Cas9 + gRNA fusion (RNPs)
plant crispr and wheat
increase grain mass of wheat
do knockdown/KO of regulatory pathways –> enable excess grain mass
3 regulatory homologs –> all have XbaI RE site
screen for mutagenesis
- amplify –> mutagenesis –> XbaI site KO’d –> will appear as larger band
- wt homologs –> digested –> smaller frags
- deliver homologs to chl –> do NOT integrate into chrom (why? recall…..)
Human Crispr examples
- leukemia + crispr corrected HSC transplants
- Amaurosis 10
Human crispr - amaurosis 10
- Amaurosis 10 (child blindness) caused by CPE290 mutation
- provide fxnal wt replacement
- 2gRNA –> CPE290 mutant excision –> HDR to replace
- tested on mice –> imperfect but at least improves vision
making transgenic mice: transfection to transgenic founder
- superovulated female –> harvest eggs
- direct injection to male pronuclei –> recombination
- zygote matures –> heterozygous mutant
- cross breed 2 mutants –> screen for homozygous mutant using PCR/South blot
- confirmational assays with western blot
transgenic mice - Modifying for protein expression (how to create a producer cell line) + What specific genes are needed?
- use lentiviral vector transduction (form of retrovirus)
- mutated virus so virion can’t replicate –> prevent lysis/harm
Important vector genes for expression
Psy gene = Allows packaging when transfected into packaging mice cell line
PPT gene = enhance transfection efficiency
WPRE = enhance GOI expression
Ensuring transgenes are integrated at specific sites in animal cells - transgenes involved for selection/identification + how the method works
Insert GOI as part of a casette –> GOI flanked by hb1/hb2, further flanked by tk1/tk2 on outsides
transfection –> selection by NeoR (aminoglycoside G418 resistance)
random crossover/recombination –> entire cassette inserted –> tk1/2 included –> express toxins
specific recombination –> partial cassette integration –> only GOI + NeoR + hb1/2 –> No tk1/2 toxins expressed
therefore:
NeoR selects for transfectants
tk1/2 presence selects against non-specific recombinants
Animal crispr - why make pharmaceuticals in milk?
Allows harvest of proteins w/o having to kill the animal –> cheaper long term + more protein yield per animal
preferrably goats/cows
cows make the most protein per L of milk, goats are cheaper to maintain
Animal crispr - transgenic milk examples
antithrombin - goats milk - integrate antithrombin gene into beta-casein gene, imperfect glycosylation to human form but retains fxn well enough for human usage regardless
Lysostaphin - transgenically expressed in cows to prevent mastitis of the udders - present in wt tissues but inactive. Mutate cow fibroblasts to activate lysostaphin expression –> transplant into host
Animal Crispr - Fish
GMO salmon to increase growth hormones –> salmon grows faster + can be grown over winters –> year round salmon supply
GMO fish to detect estrogen pollutants in water –> introduce promoter that binds estrogen analogs, use to control/activate GFP –> therefore green fish = estrogen pollution
