Conclusions
This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.
Background
Von Hippel Lindau disease is a rare genetic disorder that effects 1 in 36,000 individuals. It is characterized by tumors and cysts that grow throughout the body, called hemangioblastomas [1]. The disease is caused by mutation in the VHL gene. The VHL gene is a tumor suppressor, which regulates downstream genes that play a role in cell division, and thus mutations in the VHL gene lead to uncontrolled cell division or tumors. For many Von Hippel Lindau patients, renal cell carcinoma is the leading cause of death [2]. Therefore, I decided to focus my research on the role that the VHL gene plays in kidney development.
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Aim 1: Identify conserved amino acids in VHL responsible for kidney development.
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To identify conserved amino acids in the VHL box domain, I would align the protein sequences of all the homologs of VHL using Clustal Omega. I would than use Jalview to visualize these results. From this alignment, I will identify amino acids that are conserved across all organisms with metanephric and mesonephric systems.
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Using CRISPR, I would edit the sequence to replace the amino acid in zebrafish with the amino acid of drosophila at each point. I hypothesize that changing the amino acids would obtain mutant VHL zebrafish will demonstrate phenotypes of renal cell carcinoma like a loss of the negative regulation of cell division.
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Aim 2: Identify a small molecule that restores function in VHL mutant zebrafish with kidney tumors.
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To obtain my diversity-oriented library, I would run the structure of the VHL protein and the structure of potential ligands through Dock, which is a provides the probably of binding. Once I had obtained my diversity-oriented library, I would run a high throughput chemical screen using wildtype zebrafish, as well as my mutant VHL zebrafish from Aim 1. To do this, I would harvest the kidney tissue from each group. I would put them through the chemical screen and look for a restoration of the negative regulation of cell proliferation and apoptosis. I would want to see a restoration of levels in the mutant VHL zebrafish that was the same as the level in the wildtype. The small molecule(s) I find could be used in the future to help restore function of the VHL protein in humans.
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Aim 3: Identify novel VHL protein interactions important for kidney development.
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Future Directions
In the future, I would like to future investigate the function of VHL as a transcription factor. To do this, I would like to perform RNASeq on my wildtype and mutant VHL zebrafish. I would expect to see decreased expression of genes involved in negative regulation of cell proliferation and apoptosis.
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References
- Wong, M., Chu, Y.-H., Tan, H. L., Bessho, H., Ngeow, J., Tang, T., & Tan, M.-H. (2016). Clinical and molecular characteristics of East Asian patients with von Hippel–Lindau syndrome. Chinese Journal of Cancer, 35, 79. http://doi.org/10.1186/s40880-016-0141-z
- Von Hippel-Lindau Syndrome: Genetics Home Reference. Retrieved from < https://ghr.nlm.nih.gov/condition/von-hippel-lindau-syndrome# >
Image References
Zebrafish http://4.bp.blogspot.com/-qHe9PH1Z3Pc/UY0TJT4zRmI/AAAAAAAAAHc/kOzSdbPbHbg/s1600/zebrafish.png
Normal kidney: https://www.consumerhealthdigest.com/wp-content/uploads/2015/02/kidney-cancer.jpg
Disease kidney: https://www.consumerhealthdigest.com/wp-content/uploads/2015/02/kidney-cancer.jpg
Human: http://www.okclipart.com/Human-Clip-Art30uuwsszvs/
Mouse: http://www.clipartkid.com/cartoon-mouse-cliparts/
Rat: http://clipart-library.com/clipart/8iGEdqArT.htm
Xenopus: http://clipart-library.com/clipart/di9Kdbn5T.htm
Fly: https://upload.wikimedia.org/wikipedia/commons/thumb/6/6e/Drosophila-drawing.svg/2000px-Drosophila-drawing.svg.png
Kidney development: http://www.nature.com/nrneph/journal/v9/n3/images/nrneph.2012.290-f1.jpg
Normal and mutant kidney cells: http://www.nature.com/nrneph/journal/v11/n7/images/nrneph.2015.82-f5.jpg
Well plates: http://www.nature.com/nature/journal/v432/n7019/images/nature03197-f2.2.jpg
Normal kidney: https://www.consumerhealthdigest.com/wp-content/uploads/2015/02/kidney-cancer.jpg
Disease kidney: https://www.consumerhealthdigest.com/wp-content/uploads/2015/02/kidney-cancer.jpg
Human: http://www.okclipart.com/Human-Clip-Art30uuwsszvs/
Mouse: http://www.clipartkid.com/cartoon-mouse-cliparts/
Rat: http://clipart-library.com/clipart/8iGEdqArT.htm
Xenopus: http://clipart-library.com/clipart/di9Kdbn5T.htm
Fly: https://upload.wikimedia.org/wikipedia/commons/thumb/6/6e/Drosophila-drawing.svg/2000px-Drosophila-drawing.svg.png
Kidney development: http://www.nature.com/nrneph/journal/v9/n3/images/nrneph.2012.290-f1.jpg
Normal and mutant kidney cells: http://www.nature.com/nrneph/journal/v11/n7/images/nrneph.2015.82-f5.jpg
Well plates: http://www.nature.com/nature/journal/v432/n7019/images/nature03197-f2.2.jpg
waldman_4-27-17.pptx |
waldman_finalpresentation.pdf |
waldmanrd1.2-21-17.pdf |