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Characterization of Treacher Collin Syndrome Gene Tcof1 in Zebrafish , Danio rerio

Characterization of Treacher Collin Syndrome Gene Tcof1 in Zebrafish , Danio rerio. Annie Lee Mentors: Rita Shiang and Jim Lister. Background information of Tcof1. Human chromosome 5q32-33 Treacle Ribosomal Biogenesis Nop56p UBF. Treacher Collin Syndrome. Mutation on Tcof1

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Characterization of Treacher Collin Syndrome Gene Tcof1 in Zebrafish , Danio rerio

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  1. Characterization of Treacher Collin Syndrome Gene Tcof1 in Zebrafish, Daniorerio Annie Lee Mentors: Rita Shiang and Jim Lister

  2. Background information of Tcof1 Human chromosome 5q32-33 Treacle Ribosomal Biogenesis Nop56p UBF

  3. Treacher Collin Syndrome Mutation on Tcof1 autosomal dominant, craniofacial disorder Deficient ribosome biogenesis stabilization of p53 Apoptosis of neural crest cells

  4. Zebrafish, Danio rerio • Advantages • Disadvantage Transparent embryo Fast reproduction Development outside Knockdowns using morpholinos Less homology to human genes

  5. Previous Research • Confirm mutated Tcof1 apoptosis (P53) • Zebrafish: animal model to study TCS • Possible rescue: down-regulate p53 Uninjected Morpholino injected P53 Rescue

  6. Previous Research • Downstream genes? • P21 • cell cycle arrest • overexpressed

  7. Hypothesis • Confirm zebrafish is an efficient model to study TCS • 2. Mutated Tcof1 is involved in interrupting ribosomal biogenesis • 3. Mutated Tcof1 leads to an overexpression of p21

  8. Objective & Method • Characterize TCS using zebrafish • Confirm knockdown • Western Blotting • Rescue- TUNEL • Confirm Tcof1 mutation affects ribosome biogenesis • Real time PCR • Northern Blotting • Examine if Tcof1 mutation leads to an overexpression of p21 • Real time PCR

  9. Objective & Method • Characterize TCS using zebrafish • Confirm knockdown • Western Blotting • Rescue- TUNEL • Confirm Tcof1 mutation affects ribosome biogenesis • Real time PCR • Northern Blotting • Examine if Tcof1 mutation leads to an overexpression of p21 • Real time PCR

  10. 1)Confirm KnockdownWestern Blotting:Protein Preparation  

  11. 1. Confirm Knockdown Western Blotting

  12. Wildtype Morpholino Standard Morpholino Wildtype 260  150 100 

  13. Expected Results for Western • Nopp140 • Wildtype: dark band • Mutant: faint band

  14. 2. Confirmation of knockdown:TUNEL Preparation Apoptosis detection- labels free 3’OH end

  15. 2. Confirm RescueTUNEL A B C (A)Uninjected (B) Morpholino injected (C) Tcof1 RNA Rescue

  16. Problems: TUNEL A B (A) Uninjected (B) Morpholino injected

  17. Objective & Method • Characterize TCS using zebrafish • Confirm knockdown • Western Blotting • Rescue- TUNEL • Confirm Tcof1 mutation affects ribosome biogenesis • Real time PCR • Northern Blotting • Examine if Tcof1 mutation leads to an overexpression of p21 • Real time PCR

  18. 1. Ribosomal BiogenesisReal Time PCR

  19. 1. Ribosomal Biogenesis- Real Time PCR Uninjected Rescue-P53 Morpholino Injected Mixed Morpholino Injected Log10 (Relative Quantity) Primer z18s

  20. 2. Ribosomal Biogenesis-Northern Blotting RNA and Probe Preparation RNA Preparation    Probe Preparation

  21. 2. Ribosomal Biogenesis-Northern Blotting

  22. 2. Ribosomal Biogenesis-Northern BlottingExpected Results RNA from mutant: faint RNA from wildtype: dark

  23. Objective & Method • Characterize TCS using zebrafish • Confirm knockdown • Western Blotting • Rescue- TUNEL • Confirm Tcof1 mutation affects ribosome biogenesis • Real time PCR • Northern Blotting • Examine if Tcof1 mutation leads to an overexpression of p21 • Real time PCR

  24. 1. P21 gene expression- Real Time PCR Log10 (Relative Quantity) Mixed Morpholino Injected Morpholino Injected Rescue-p53 Uninjected Primer zCdkn1a

  25. Some Conclusions • Zebrafish is an efficient model to study Treacher Collin Syndrome • Tcof1 involvement in ribosomal biogenesis is still inconclusive • p21 is overexpressed when there is mutation with Tcof1

  26. Further Research • Confirm that mutation of Tcof1 causes deficiency in ribosomal biogenesis • Ways to treat TCS • Rescue with p21 • Down-regulate p21

  27. Reference [1] Dixon, J., Brakebusch, C., Fassler, R., & Dixon, M. J. (2000). Increased levels of apoptosis in the prefusion neural folds underlie the craniofacial disorder, treacher collins syndrome. Human Molecular Genetics, 9(10), 1473-1480. [2] Gonzales, B., Henning, D., So, R. B., Dixon, J., Dixon, M. J., & Valdez, B. C. (2005). The treacher collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation. Human Molecular Genetics, 14(14), 2035-2043. [3] Hayano, T., Yanagida, M., Yamauchi, Y., Shinkawa, T., Isobe, T., & Takahashi, N. (2003). Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. possible link between Nop56p and the nucleolar protein treacle responsible for treacher collins syndrome. The Journal of Biological Chemistry, 278(36), 34309-34319. [4] Holster, T. M. "A new model for Treacher Collins Syndrome: characterizing the zebrafish tcof1 ortholog." Unpublished. [5] Knecht, A., & Bronner-Fraser, M. (2002). Induction of neural crest cell: a multigene process, Nature Reviews Genetics. 3(819) 453-461 [6]Meier, U. T., & Blobel, G. (1992). Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell, 70(1), 127-138. [7] Mogass, M., York, T. P., Li, L., Rujirabanjerd, S., & Shiang, R. (2004). Genomewide analysis of gene expression associated with Tcof1 in mouse neuroblastoma. Biochemical and Biophysical Research Communications, 325(1), 124-132. [8]Patel, A. D. "Characterization of zebrafish models of Treacher Collins syndrome." Unpublished (2008). [9] Splendore, A., Jabs, E. W., Felix, T. M., & Passos-Bueno, M. R. (2003). Parental origin of mutations in sporadic cases of treacher collins syndrome. European Journal of Human Genetics : EJHG, 11(9), 718-722. [10] The Treacher Collins Syndrome Collaborative Group, Positional cloning of a gene involved in the pathogenesis of Treacher Collins syndrome, Nat. Genet. 12(1996) 139-136.

  28. Acknowledgements Rita Shiang Sami Omer Balsam Jim Lister Brendon Leyla

  29. Questions?

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