13. Choose one of the two following case scenarios to work on. You MUST stay wit
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13. Choose one of the two following case scenarios to work on. You MUST stay within your budget for examining your question. (40 pts.) The flu virus has become extremely dangerous this flu season. You are interested in determining how one protein of the flu virus impacts eukaryotic cells. You have been given $1000 to examine this protein. Design your experiment from beginning to end. A) Transposon movement is suspected to be a cause of remission for certain cancers. You want to determine if this is true. Design an experiment to test is transposon movement results in remission of cancer/ tumors. You have $2000.00 to test your hypothesis. B) State clearly the hypothesis that you are testing with your experimental design. (S pts.) Describe a reasonable method for approaching this project including preparing your template for cloning, type of cloning used, vector, and why you chose this particular methodology vs. others. (20 pts.) Also include a budget (using an Excel spreadsheet) to show supply cost for doing this project. This includes enzymes, enzyme mixes, cells, and vectors needed. Assume you have tips, micropipettes, and all other molecular equipment that is currently available in the genetics lab for you to use. Go to www.fishersci.com to get some pricing on your needed supplies and generate your budget in an Excel spreadsheet. (10 pts.) Describe any potential 'pitfalls' to your experimental design based on funding or other issues and how you would resolve these problems if you had unlimited funding. If you do not see any potential pitfalls in your methodology, feel free to state this but you must also then address how your design is essentially foolproof.(5 pts.) 1. 2. 3. 4. Please cite any sources used (other than vendor websites) as appropriate in-text and at the end of your answer.Explanation / Answer
A. Impact of flu protein on eukaryotic cells:-
Influenza infection stimulates the host innate immune system by inducing interferons (IFNs).
Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function.
To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs.
Experimental design:-
HeLa cells were grown as.
- monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U of penicillin/ml, and 100 g of streptomycin sulfate/ml at 37°C.
When the cells were approximately 80% confluent, supernatant was removed and replaced with either medium alone (Dulbecco's modified Eagle's medium with 2% calf serum), medium containing untreated influenza virus or medium containing inactivated virus.
Viral attachment was carried out at 4°C for 45 min with gentle agitation. Cells were then incubated at 37°C for 4 or 8 h. At the indicated times, medium was removed, the cells were harvested, and the total RNA was extracted using the method of Chomczynski and Sacchi.
Inactivation procedures and agglutination assay.
Heat inactivation of influenza virus stocks was performed by incubating the virus stock at 56°C for 90 min.
UV inactivation of influenza virus also performed.
Optimal inactivation was determined by methionine-cysteine labeling of infected cells to be 80 mJ.
To determine if the viral hemagglutinin (HA) protein was still active after inactivation procedure, agglutination assays Effect of inactivation of influenza virus on the synthesis of host cell proteins and viral hemagglutination titers.
(A) Protein extracts from mock-infected cells, cells infected with heat-inactivated virus and cells infected ...
Analysis of cellular and viral protein synthesis.
The extent of influenza virus infection and the effectiveness of the inactivation procedure were monitored by examining host cell protein synthesis after infection. virus-infected HeLa cells were labeled at the indicated times with methionine-cysteine, and protein extracts were examined on 14% sodium dodecyl sulfate (SDS)-polyacrylamide gels.
# RNA isolation, first-strand cDNA synthesis, and Northern blot analysis.
a virus-infected HeLa cells, 2.5 ng of green fluorescent protein poly(A)+ RNA, 8 pmol of anchored dT primer, and 1 g of random nonamers were combined in a 10.5-l reaction volume.
The solution was heated to 70°C for 10 min, chilled briefly on ice,
Fluorescently labeled first-strand cDNAs were concentrated by drying awas a set of 384 selected cDNAs that were spotted on every slide.
This set contained four influenza virus genes used as positive controls,
nonhuman genes used as negative controls.
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