Transgenic Plants
More effort is currently directed toward making plants not higher yielding but resistant to: herbicides, insects, fungal infections, and viruses.
Herbicide resistance
Herbicide glyphosate (in Round-Up) blocks EPSPS (5-enoylpyruvoylshikimate-3- phosphate synthase; encoded by aroA gene) in the common pathway for aromatic amino acids; deadly to plants and most bacteria but safe for humans and other animals because humans can't synthesize aromatic a.a. so don't have aroA gene
Identified bacteria that were resistant to glyphosate; cloned the mutant aroA gene; used it to transform plant cells in culture and make resistant tomatoes, potatoes, petunias, etc.
(Incidentally, aroA mutants of Salmonella gallinarum (causes fowl typhoid) that can't synthesize EPSPS are being used as attenuated live vaccines in chickens. The mutant strains grow if supplied with aminobenzoic acid but don't grow in the chickens because aminobenzoic acid is not readily available in vertebrate tissue. However, the mutant strains survive in the chicken long enough for the chicken to mount an immune response.)
Insect resistance
Bacillus thuringiensis produces an insecticide that kills a number of caterpillars. But when cloned into plant, not much toxin was produced so:
1) Decreased size of toxin gene from 1156 a.a. to 650 a.a. at N-terminus (still active)
2) Put under control of a strong constitutive promoter (CMV-cauliflower mosaic virus promoter was used)
3) Changed codons so that codon usage was more plant-like (Example of different codon usage: AAA and AAG both code for lysine. E. coli uses AAA 75% of the time but Rhodobacter uses AAG 75% of the time)
Anthrax vaccine
Bacillus anthracis is the causitive agent of anthrax
Need to use the PA (protective antigen) protein as an anthrax vaccine
Cloned PA gene into tobacco plants
Cloned into chloroplast genome: 1) hundreds of chloroplasts per cell so higher protein production; 2) chloroplast DNA is not present in pollen so less chance of transferring PA gene to unintended organism
Transforming plant cells
2 ways: 1) plasmid or 2) particle bombardment
Transformation using Ti Plasmid
Agrobacterium has a Ti (tumor-inducing) plasmid. Agrobacterium is attracted by hormones released from an injured plant; infects plant and transfers part of Ti plasmid (T-DNA) to plant cell; T-DNA (tumor-DNA) is integrated into plant nuclear DNA; causes crown gall tumor to form
T-DNA contains genes that direct the synthesis of plant hormones auxin (makes plant cells bigger) and cytokinin (makes plant cells divide); result of rapid growth and division is a tumor
T-DNA also contains gene for synthesis of opines, nutrient molecules that can only be utilized by bacteria that posses a Ti plasmid. Opine utilization genes are located on the part of the Ti plasmid that aren't transferred to the plant. So plant now makes nutrients that only Agrobacterium can use.
Clone an ori for E. coli into the Ti plasmid; now can work in E. coli. Now clone useful gene into Ti plasmid by replacing genes in the T-DNA (but keeping the left and right borders of the T-DNA intact). Then put engineered Ti plasmid back into Agrobacterium and allow it to infect plant cells in culture.
Ti plasmids don't work on monocots (grasses, grains, conifers) so use bombardment.
Transgenic Animals
Production of Transgenic Animals by Microinjection
1) Clone a transgene (useful gene to be put into animal)
2) Inject transgene into male pronucleus of fertilized egg (larger than female pronucl.)
3) Implant engineered egg into female animal (surrogate)
4) Assess progeny for stable integration of transgene
Founder mice - those that have the transgene integrated; are heterozygous for transgene because have only one copy of the transgene
Mate two founder mice and will get 1/4 progeny with no transgene, 1/2 progeny heterozygous for transgene, and 1/4 progeny homozygous for transgene (most useful)
Knockout Mice for Studying Gene Function
Disrupt gene by inserting a gene cassette. Select for disruption (in E. coli) by antibiotic selection because of antibiotic resistance gene carried on gene cassette. Use disrupted gene to transform fertilized mouse eggs. Can produce founder mice with only one copy of the mutant gene or mate the founder mice to get a double knockout with both copies of the gene mutated. Double knockouts won't survive if gene is essential. A more recent reporter is the GFP gene that encodes green fluorescent protein that glows in black light. First animal transformed with GFP was mice, now have GFP rabbits, chickens and pigs.
Particle Bombardment
Use particle gun to fire gold (inert and non-toxic) particles carrying DNA into cells. Use reporter genes to detect presence of desired DNA
Reporter gene npt - neomycin phosphotransferase - confers neomycin resistance
Reporter gene luc - luciferase - emits light in presence of luciferin; was used to transform tobacco
Can bombard chunks of animal organ (e.g. brain) and reimplant into animal. Rat fetal brain tissue was bombarded with gene for tyrosine hydroxylase (involved in the synthesis of dopamine), reimplanted into adult rat and gene was expressed. May be used as gene therapy for Parkinson's disease.
Transgenic cows for drugs that only express new gene in the mammary gland so the protein comes out in the milk. Transgenic goats have been generated that make recombinant tissue plasminogen activator to dissolve blood clots.
To clone an animal: Remove nucleus from donor. Need a totipotent cell that can give rise to any cell type (as found in embryonic stem cells). But can also make a cell totipotent by starving the cell so that it stops dividing. Demethylation may take place. The resting nucleus is then removed from the starved cell and inserted into an enucleated egg cell. This cell is then implanted into the uterus of a surrogate female.
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Last updated: 9-April-98 / laa