BSCI222 – Lecture 21 (11/19/13)- Genetic mutations in eukaryoteso Genetic engineering in plants: Basically taken over US agriculture GMO crops are differently accepted around the world Technology: have to get the DNA sequences that you’re interested in into the plant cells. To do that, use system derived from natural agrobacterium (which enters wounds in the root system, creates a nodule for itself to live in. A lot like F plasmids, in that this plasmid encodes the genes necessary to establish the transfer of DNA into the host cell for recombination into the plant chromosomes.) Take foreign DNA  put into plasmid  put plasmid into bacterium that already has the tumor-inducing plasmid from agrobacterium  has all the genes necessary for the transfer. Have plasmid vector, the helper Ti plasmid, and bacterial chromosome. First commercially viable: BT (bacillus thuringiensis) transgenic corn. People were trying to solve problem from European corn borer (moth, infects the corn ears). Widely-used pesticide, crystal toxin proteins, naturally-occurring soil bacterium. Take BT gene  clone into plasmid hooked up to antibiotic resistance gene (track and select cells that have it)  put into expression vector (not just interested in cloning the DNA and inserting, want it to have a strong promoter and be expressed)  put into the agrobacterium with the TI plasmid, creating a hybrid plasmid to increase frequency of transfer into plant cell  whole construct is put intothe corn plants. Benefits: high levels of expression of a toxin within a plant (high dosage of pesticide), toxin expression is contained within the plant (only the insects eating it are affected, others in the environment aren’t), during the first 10 years of BT crop use, insecticide use fell by 35.6 million kg. HT plants: herbicide-tolerant, herbicide won’t kill the crop, will kill everything else. Can stack both together in a plant. Almost everything is from GMO corn today. Potential problems with this technology: insects might evolve resistance tothe BT (European borer moths, raised on the crystalline toxin, resistance quickly grew). In some parts of the world, the crop is no longer effective at killing pests. Pollen from BT corn might be toxic to Monarch butterflies(raised on milkweed, milkweed + non-BT corn, and milkweed + BT corn pollen)  only about half survived after a few days with the BT corn pollen. BUT, the pollen wasn’t pure, was about 43% plant debris. Little BT actually expressed in the pollen, but still. Farmer lost lawsuit to Monsanto because neighbor’s Monsanto pollen drifted over and pollinatedhis crops, re-planted seeds. Finally, certain crystalline toxins from certainBT strains can kill mammalian cells (matching between the toxin protein and various receptors on the human cells, needs to be studied more). o Transgenic mice:  Important tool for biomedical research Genome size similar to us, essentially same genes.  Simplest technique: grow many copies of the interested gene, inject it into nucleus right after fertilization, and hoping that it somehow gets randomly incorporated. Not very high efficiency, have to sort through all the babies looking for the occasional individuals that have it. Random integration, can’t control the site in the genome where it is placed. Can then breed the transgenic ones until your get mice that are homozygous for the gene. More difficult if trying to knock-out a gene that is already in the mouse genome. First, have to clone a copy of the gene you want to KO. Then, putantibiotic resistance gene into the middle of it, to knock it out (interrupted the reading, and now have a marker to track the gene and select cells). Also attach a copy of the thymine kinase gene (tk), susceptible to gancylovir. Put the whole construct into mouse embryonic stem cells in culture. To find the ones that pick up the construct, use neomycin (antibiotic resistance is neo+). Looking to get recombination in the stem cells between the construct and the target gene. Have a lot of homology, which drives the recombination, and hopefully transfer in the disruptive neo+ block into the middle of the target gene, by homologous recombination (not random, trying to control where the integration happens). After transfer, looking for the recombinant ones, which have theneomycin resistance but not the thymine kinase (not the ones that are just carrying the plasmid, which has tk attached to it. The recombinant chromosome is neo+ and tk-). The modified stem cells can be put into a mouse embryo, into a pseudo-pregnant mouse, to get embryos out of it. The embryonic stem cells are from black strain of mice, while injected into cells from white strain, and thus the pigmentation of the progeny tells you which animals have successfully taken up the cell. Then, have to breed them to produce homozygosity (completely black mouse), knocked out gene.- Random integration would give you both the neo+ and the tk+- What about genetic therapies for humans? None of this technology is attractive for fixing an adult human. Could presumably modify offspring, but it’s expensive and ethically issue-prone. Most of the focus has been on, how can we deliver genetic constructs to adult humans and have an effective transfer in order to affect the phenotype?o RNA interference: one of the more promising. Put construct with a copy of the gene that you want to KD expression off, with promoter on both sides  double stranded RNA  Dicer chops it up  RNA-induced silencing complex (silence expression of RNAs from that gene). Example: KD cholesterol levels (ApoB, KD  fewer particles in bloodstreams). Has been done in monkeys; just put theRNAI into the bloodstream, cholesterol went down 60-70 points, very effective. Want to make this permanent, not using IV every other day, want constant expression. How to deliver those constructs? Can’t go and inject it into lots of different cells, have to figure out some viral method for getting it into the cells. A lot of methods have been tried; adenovirus looks the most promising. Transfers the construct into non-dividing cells, but can cause a strong immune reaction. Decade ago, clinical trial for gene therapy, Jesse Gelsinger was a volunteer, had some medical complications but was still admitted as a participant in the trial, wasinjected with an adenovirus vector carrying a gene, and very quickly had a strong immune