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CHAPTER 21 1. Transgenic mice can be created by cloning the transgene into a retrovirus and infecting an early-stage embryo with the virus, by microinjection of DNA into the sperm pronucleus of an embryo or by injecting transgenic embryonic stem cells into an earlystage embryo. The embryo is then implanted in the uterus of a surrogate mother, where, if the DNA was incorporated into the embryo, a transgenic mouse develops. The retrovirus method can be used only if the transgene is small (≈8 kb). A second disadvantage of this system is that extra viral genomic DNA may also be incorporated into the host's chromosome. On the other hand, lentiviral vectors are not limited to smaller transgenes and can infect non-dividing cells. The microinjection technique is the one typically used to create transgenic mice. A donor female is superovulated, mated and then killed to harvest the eggs. The male pronucleus from the sperm is larger than the female pronucleus and can be seen under a dissecting microscope. The linear DNA is then injected into the male pronucleus. The disadvantage of this system is that it requires highly trained technicians, and even then, only 5% of the eggs develop into transgenic mice. The method is highly inefficient at each step and even if the DNA is integrated into the embryo, there is a chance that due to positionality and copy number, the offspring will still not be useful. The embryonic injection method utilizes genetically engineered stem cells (which are easier to engineer than full embryos and eliminates the problem of random integration of the transgene into the host chromosome). The chimeric mice are then breed for a transgenic homozygote. 2. Positive/Negative selection works by a) killing off the cells that do not have vector DNA anywhere in their genome, and b) killing off all of the transformed cells that did not occur by homologous recombination and therefore do not have the transgene at the desired location. The first case is considered positive selection and involves the insertion of the gene for neomycin resistance. After the transfection of cells using the embryonic stem cell method, the non-transformed cells are killed off by exposure to neomycin. Only the transformed cells, with viral DNA somewhere in the chromosome are resistant to the neomycin treatment. The second case is considered negative selection and is possible because the thymidine kinase gene which is present in the injected DNA produces a lethal toxin when treated with the drug ganciclovir. The thymidine kinase gene is present in the vector DNA, but not within the “fake gene” region designed to mimic the site on the chromosome where you want the transgene to insert. If the transfection occurred by chromosomal rearrangement, then only the knockout gene fragment of the DNA transferred to the chromosome, specifically knocking out the gene of interest and carrying the neomycin resistance. If the transformation occurred by random insertion, then the whole DNA insert, including the thymidine kinase gene, is present somewhere in the chromosome, but not at the desired position. 3. Knockout mice are mice with a specific gene that is completely nonfunctional. Gene expression has been abolished using the embryonic stem cell method of transgenesis where a dysfunctional copy of the gene of interest replaces the functional gene through homologous recombination. These lines are established to provided researchers with a living organism without the functioning gene. By observing the mutant phenotype, scientists are able to draw conclusions about the exact function of the gene in the wild type animal. 4. Knockdown mice are mice where gene expression is decreased using RNAi. The mouse has a mutant phenotype not because there is anything wrong with the wild type gene, but because the RNA is eliminated preventing the production of protein. Knockdown mice are made by cloning a portion of the gene of interest, followed by a spacer, followed by the inverted portion of the gene of interest into a vector. The sequence is arranged so that the RNA transcript will form a double-stranded hairpin. This vector DNA is then added to the mouse embryo via microinjection or lentiviral vector. RNAi (Dicer and RISC degrade the dsRNA and produce microRNAs which then bind to the endogenous mRNA and mark them for degradation). 6. The advantages of using transgenic mice as a model system for human diseases are that you can test out potential drugs and therapeutic agents on the mice before the treatments ever go to humans. The etiology of complex diseases can also be examined in mice to improve knowledge of how the disease functions and progresses. The main disadvantage is that mice are not humans and there is no guarantee that a drug will work the same on a mouse as it does on a human, or that a disease will affect mice the same as it does humans (e.g. cystic fibrosis in humans generally manifests itself as a lung disease, but in mice the dominating symptom is an intestinal phenotype). 7. A mouse system was developed for understanding Alzheimer disease in humans. Alzheimer disease is a degenerative disorder of the brain where plaques develop at the synaptic junctions of nerves and neurofibrillary tangles develop in the cell body. The proteins produced by APP genes are the primary component of the senile plaques. Mouse models were produced with altered APP genes. A brain-specific promoter was fused to APP cDNA (with the introns added back). Overexpression of the PDAPP construct in elderly mice produces the symptoms of Alzheimers (memory loss, neuron death and senile plaques). The mice do not develop neurofibrillary tangles, though, so that is either caused by a second gene or is specific to the human Alzheimer's phenotype. Transgenic mice have also been produced with BACE1 knocked out. BACE1 is a protease that cleaves APP. Transgenic mice that produce APP but do not have BACE1 and do not develop senile plaques. 10. The mammary gland has been used as a bioreactor for the production of pharmaceutical proteins. If transgenic animals are engineered with a mammary gland promoter and the signal sequences for the secretory pathway, large quantities of a drug can be purified out of the milk and sold for human consumption. The cystic fibrosis transmembrane regulator (CFTR) protein, antithrombin III, growth hormone, and insulin are just a few therapeutic proteins that are being expressed in transgenic animals. Production of the protein in the mammary glands is particularly desirable since it simplifies the collection and purification of the target protein. 13. Enviropigs are pigs that have been genetically engineered to carry a bacterial phytase gene that allows for the digestion of phytate. The primary storage form of phosphorous used by plants is phytate, but only ruminants are able to utilize the phosphorous stored in this form. Enviropigs are able to convert phytate to inositol 2-monophosphate or inositol, decreasing the need for phosphorous dietary supplements and lowering the phosphorous concentration excreted into the environment. 15. Transgenic salmon have been produced with enhanced growth rates and better stress and disease tolerance. Inserting an “all-salmon” growth hormone makes transgenic fish that grow 3-5 times larger than wild type fish in a shorter period of time. Decreasing the time it takes to produce full grown salmon will theoretically decrease production costs and pollution, improving aquaculture. CHAPTER 5 1. The sequence with accession number NM_000492.3 is the mRNA for Homo sapiens cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) (CFTR). 2. This protein is 100% similar to several huntington disease protein sequences in the database. 10. In humans, the number of proteins outnumbers the number of genes because 60-70% of human genes undergo some type of alternative splicing. This means that 30,000 genes can produce approximately 85,000 proteins. 11. The principal feature of 2D PAGE is that proteins are separated first by one characteristic and then by another (i.e. isoelectric focusing point and size). 2D PAGE has high resolving power and is used to isolate individual proteins. Proteins are separated by isoelectric focusing point by applying the proteins to a pH gradient gel and then introducing an electrical current. When the protein has arrived at the position in the gradient where the molecule has no net charge, then it will stop. If the second dimension is size, the proteins are again separated on a gel with electricity. When proteins are denatured to their primary structure, SDS ions (sodium dodecyl sulfate) surround the proteins, masking the inherent charge of the protein and providing their own charge (which is proportional to size since the number of SDS molecules that can associate with a protein is determined by the size of the protein). The electrical current then causes proteins to migrate through the gel at a rate the is proportional to the charge provided by the SDS. The smaller proteins are subjected to less electrical force and less drag through the gel while larger proteins are subjected to more electrical force and more drag. In a polyacrylamide gel, though, the force of drag cancels out the difference in electrical force so that smaller proteins migrate faster than the larger ones. 14. The protein is human serum albumin. MatrixScience.com also identifies the protein as human serum albumin (Top Score: 113 for ALBU_HUMAN, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2).