Midterm 2B

Question 1

Vitamin A and Rice initial + more recent transgenic mods

Answer 1

• 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

Question 2

Vit B9 (folate) deficiency in rice

Answer 2

• 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

Question 3

tomato ripening - what genes, what do they do, how was flavrsavr tomato modified?

Answer 3

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

Question 4

Potatoes - transgenic starch reduction?

Answer 4

amylose + starch branching enzymes (SBE) = amylopectin
reduce amylopectin presence using RNAi of SBEs –> potatoe cells less dmaaged after sustained freeze/thaw cycles

Question 5

transgenic trees for pulp production

Answer 5

• 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

Question 6

Why use plants to produce things?

Answer 6

• 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

Question 7

transgenic plants - viral vectors - examples + how the virusses were modded

Answer 7

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

Question 8

Transgenic plants - Why not integrate the GOI into chromosomes?

Answer 8

• 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

Question 9

plantibodies

Answer 9

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

Question 10

edible vaccines - example + method

Answer 10

• 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

Question 11

modifying cholera toxin for wider vaccinations

Answer 11

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

Question 12

Ecoli modified to use HDR - What plasmids involved?

Answer 12

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)

Question 13

Ecoli modified to use HDR - confirmational testing - the premise

Answer 13

• 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

Question 14

Ecoli modified to use HDR - confirmational testing - the results/interpretations on mutation efficiency

Answer 14

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

Question 15

Plant crispr - how and why? What molecule(s) used to specifically faciilitate this transfection?

Answer 15

agrobacterium transfection
delivery into chl w/o integration into chromosome (DNA free editting)
use preassembled Cas9 + gRNA fusion (RNPs)

Question 16

plant crispr and wheat

Answer 16

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…..)

Question 17

Human Crispr examples

Answer 17

• leukemia + crispr corrected HSC transplants
• Amaurosis 10

Question 18

Human crispr - amaurosis 10

Answer 18

• 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

Question 19

making transgenic mice: transfection to transgenic founder

Answer 19

• 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

Question 20

transgenic mice - Modifying for protein expression (how to create a producer cell line) + What specific genes are needed?

Answer 20

• 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

Question 21

Ensuring transgenes are integrated at specific sites in animal cells - transgenes involved for selection/identification + how the method works

Answer 21

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

Question 22

Animal crispr - why make pharmaceuticals in milk?

Answer 22

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

Question 23

Animal crispr - transgenic milk examples

Answer 23

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

Question 24

Animal Crispr - Fish

Answer 24

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

Question 25

BSE - How it started

Answer 25

Sheep have prions –> “Scrapies” –> safe for human consumption (no effects)
scrapies comes from precursor protein prion (PRP gene) aggregating as stacks of beta sheets
feed scrapies sheep to cows to supplement food –> treat sheeps meat with high T –> safe for cows
lazy –> poor T treatments –> cows infected with prions –> prion cascade –> BSE

Question 26

BSE - how to fix with transgenic sheep

Answer 26

eggs -> microingection -> mutation of sheep’s PRP gene (disables ability for prions to aggregate)