HC70A quiz 7 - Name Marissa Lara Group HC70A SAS70A WINTER...

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Name: Marissa Lara Group HC70A & SAS70A WINTER 2016 PROFESSOR BOB GOLDBERG & JOHN HARADA QUIZ #7 DUE: Monday, February 29 at 5pm in Terasaki 4121. Follow Quiz #1 Guidelines QUESTION ONE – 15,000 points You are the Head of the Department of Oncology at the UCLA Medical Center and became aware of a four-year-old child with leukemia (a bone marrow cancer) who was not responding to traditional chemotherapy treatment. As a last ditch effort to save the child, you decided to use gene therapy in order to engineer the child’s bone marrow cells with a tumor necrosis factor gene (TNF) to enhance the ability of these cells to destroy the cancerous white blood cells. Note: Tumor necrosis factor is a protein that has the ability to induce cancer-cell death. Starting with (1) a “wild-type” retrovirus, (2) purified necrosis factor mRNA, (3) a bone- marrow-specific gene “switch”, (4) a retrovirus packaging cell line, (5) pBR322 plasmid (Tet R , Amp R ), (6) E. coli HB101 cells (Tet S , Amp S ), and (7) all the reagents, equipment, enzymes, and vectors required to clone DNA fragments and carry out human gene therapy studies, OUTLINE conceptually how you would engineer the child’s bone marrow cells to produce tumor necrosis factor protein. 1. isolate hematopoietic stem cells from patient’s blood 2. grow the patient’s cells in culture 3. remove viral RNA from the wild type provirus and form 2 RNA strands: 1 from the LTR sequences, PSI packaging element, the desired gene, the bone marrow specific gene switch at the front to ensure the protein is expressed specifically in the bone marrow, essentially disable the “wild type” retrovirus by removing the GAG, POL, and ENV so that these genes are no longer produced and thus the infection is no longer produced so the retroviral vector is “safe” and the other RNA from the normal retroviral genome except only removing the PSI element so that the virus cannot be packaged and thus cannot spread 4. use reverse transcriptase to convert RNA into DNA 5. use terminal transferase and DNA ligase to form a recombinant construct 6. introduce the wild type retrovirus into the cell packaging line to create more of the virus, but they are not contagious because they are disabled 7. reverse transcriptase enzyme is used in the virus to transform viral RNA genome into double stranded DNA 8. the packaging cell line replicates the viral DNA and produces many viruses and packages the packageable therapeutic gene and produces safe vectors 9.cleave pBR322 plasmid in the middle of the Amp gene and provirus with the same restriction enzyme and form a recombinant plasmids mixing the provirus DNA, plasmid DNA, bone marrow specific gene switch at the front, and terminator at the end through use of terminal transferase, annealing, and sealing with DNA ligase Make the E. Coli cells permeable by exposing to calcium chloride
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