Genetic Engineering

Paper 1:
Assessment of the nutritional values of genetically modified wheat, corn and tomato crops:
Potential problems of GM food: allergenicity, toxicity, cross contamination of antibiotic resistance, carcinogenicity, alteration in nutritional value.
GMO is only safe if the nutritional value and its toxicity is comparable to natural foods.
Tested species:
• Transgenic wheat containing tobacco rab1 gene
• Transgenic BT corn containing Bacillus thuringiensis Cry gene (toxin)
• Transgenic tomato containing Agrobacterium rhizogenes rolD gene

Tested compounds: Vitamin C, carotenoid, antioxidants, minerals, phenols, fatty acids
Results: cross contamination was less than 1% in all wheat and maize : no significant difference. In tomato samples however, antioxidant activity and naringenin content was lower than non-transgenic control plants compared to the GMO tomato. Nutritionally all three were similar to the conventional fruits in the market.

Paper 2:
Creating disease resistance cotton
Gene encoding D4E1 peptide creates resistance to alfa-toxigenic fungus A.flavus in cotton. Also done in tobacco, potato, rice and poplar tress.

Mechanism of action:
• Inhibits cell plate formation in cell division (B-D glucan synthase)
• Inhibits chitin synthesis
• Leakage of minerals from cells (cecropin B)

Advantages of using synthetic peptide in creating transgenic plants:
• It has more target specificity and more efficacy at lower concentrations
• Can affect other microbial and fungal pathogens as well as fungus A.flavus

Disadvantages of using synthetic peptide:
• It can be harmful for plants and humans
• Due to post-transcriptional effects, resistance to the disease disappears.

Paper 3:
Restoring a maize root signal that attracts insect-killing nematodes
The western corn rootworm (WCR) is the most destructive pest of maize in north America. Developing hybrids with higher level of native plant resistance to WCR is a sustainable alternative to transgenic approaches.
When attacked by insects, plants emit compounds that attract natural enemies of the insects. So these signals can be manipulated to improve crop protection.

Insect-damaged maize roots emit this type of signal (B-caryophyllene) which attracts nematodes that kill WCR worm. But due to breeding techniques most maize in America lost their ability to emit this signal. A non-emitting maize is then transformed with B-caryophyllene synthase gene (tps6) from oregano.

Results: these plants had less root damage and had 60% fewer adult beetles.

Paper 4:
Suppression of prion protein in livestock by RNA interference:
Mad Cow Disease: neurodegenerative disease:
• Normal cellular prion protein : PrPc.
• Prions: PrPSc: self-replicating protease-resistant form of prion protein
Method:
• RNAi used for silencing expression of prion protein PrP in mice and livestock
• Adapt lentivector with anti-PrP shRNAs to produce transgenic goats and cattle
Results:
• In large-animal system, lentiviral delivery of shRNAs targeting specific genes is effective to reduce expression of protein in vivo.
• shRNAs used to generate stable cell line
• a cloned transgenic goat fetus drastically reduced expression of PrP

Paper 5:
Fungus modified to express a scorpion neurotoxin:
• Problems with insecticides: toxicity, resistance, contamination.
• Better insecticide needs to be fast, effective, safe, easy, cheap
• Why use fungi? It’s easy to grow, highly selective, storable spores, kills host, safe?
• Faster death, few spores needed, selective, kills when injected.
• Application: field sprays, bednets


Paper 6:
Transgenic animals:
Applications:
• Therapeutic proteins in milk
• Knocking out antigens from pig organs for transplants
• Gene therapy

Technologies:
1. Mixing DNA with sperm. Injecting sperm to eggs. E.g: albino Xenopus expressing GFP in the eye, with eye specific (Opsin) promoter
2. Microinjection: Getting a fertilized egg from a superovulated female with small female pronocleus and large male pronucleus. Holding the fertilized egg with a holding pipette, injecting the transgene in the large male pronucleus with the injecting pipette. Implant it in a female : less than 5% success rate in mice. Random integration.
3. Stem cell culture: Inner cell mass from a donor blastocyst is cultured to give ES cells (embryonic stem cells). Then the culture is transfected by a transgene. The culture is then enriched for transfected ES cells. This culture is then microinjected into a recipient blastocyst which is then implanted to pseudopregnant female. Works well in mice, but not for all species. Uses homologous recombination and site-specific integration. E.g: birds: blastoderm cells are isolated from the yolk, cultured, transfected by transgene, inject the transfected blastoderm cells into subgerminal space of the irradiated blastoderm of the yolk, place it into a chimera chicken. E.g: chimeric mouse, chimera because blastocyst cells that developed into this mouse were derived from two different individuals.
4. Retrovirus- RNA genomes: 8-cell embryo is taken from a donor female. Retrovirus containing a transgene infects the embryo and is then implanted in a recipient female. Virus-encoded reverse transcriptase makes dsDNA copy which is then inserted into the host genome (provirus). Can insert small 8kb fragments. Contamination risk. Rarely used in commercial products.
5. Gene knockouts: isolate gene of interest eg. CFTR gene of mice, clone into vector with selectable markers, introduce mutation of interest like changing amino acids, or making deletions. Inject or electroporate it into embryonic cell culture cells, where the mutated gene integrates with HR. grow cells in selective conditions, inject cells of interest into developing embryo and implant into surrogate. Chimeric offspring should result. Useful for producing models for diseases or mutation. The goal is to create a mutation of a normally present gene.
6. Gene targeting by homologous recombination: Insertion vector method: the introduced vector DNA is cut at a unique site within a sequence which is identical or closely related to part of a chromosomal gene. HR occurs, leading to integration of the entire vector sequence including marker gene.
7. Gene targeting by homologous recombination: Replacement vector method: The marker gene is contained within the sequence homologous to the endogenous gene and the vector is cut at a unique location outside the homologous sequence. A double recombination or gene conversion results in replacement of internal sequences within the chromosomal gene by homologous sequences from the vector including the marker gene.