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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
Characterization of Treacher Collin Syndrome Gene Tcof1 in Zebrafish, Daniorerio

Annie Lee

Mentors: Rita Shiang and Jim Lister

background information of tcof1
Background information of Tcof1

Human chromosome 5q32-33

Treacle

Ribosomal Biogenesis

Nop56p

UBF

treacher collin syndrome
Treacher Collin Syndrome

Mutation on Tcof1

autosomal dominant, craniofacial disorder

Deficient ribosome biogenesis stabilization of p53

Apoptosis of neural crest cells

zebrafish danio rerio
Zebrafish, Danio rerio
  • Advantages
  • Disadvantage

Transparent embryo

Fast reproduction

Development outside

Knockdowns using morpholinos

Less homology to human genes

previous research
Previous Research
  • Confirm mutated Tcof1 apoptosis (P53)
  • Zebrafish: animal model to study TCS
  • Possible rescue: down-regulate p53

Uninjected

Morpholino injected

P53 Rescue

previous research6
Previous Research
  • Downstream genes?
    • P21
      • cell cycle arrest
      • overexpressed
hypothesis
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
objective method
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
objective method9
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
slide12

Wildtype

Morpholino

Standard

Morpholino

Wildtype

260 

150

100 

expected results for western
Expected Results for Western
  • Nopp140
    • Wildtype: dark band
    • Mutant: faint band
2 confirmation of knockdown tunel preparation
2. Confirmation of knockdown:TUNEL Preparation

Apoptosis detection- labels free 3’OH end

2 confirm rescue tunel
2. Confirm RescueTUNEL

A

B

C

(A)Uninjected (B) Morpholino injected (C) Tcof1 RNA Rescue

problems tunel
Problems: TUNEL

A

B

(A) Uninjected (B) Morpholino injected

objective method17
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
1 ribosomal biogenesis real time pcr19
1. Ribosomal Biogenesis- Real Time PCR

Uninjected

Rescue-P53

Morpholino Injected

Mixed Morpholino Injected

Log10 (Relative Quantity)

Primer z18s

2 ribosomal biogenesis northern blotting rna and probe preparation
2. Ribosomal Biogenesis-Northern Blotting RNA and Probe Preparation

RNA Preparation

Probe Preparation

2 ribosomal biogenesis northern blotting expected results
2. Ribosomal Biogenesis-Northern BlottingExpected Results

RNA from mutant: faint

RNA from wildtype: dark

objective method23
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
1 p21 gene expression real time pcr
1. P21 gene expression- Real Time PCR

Log10 (Relative Quantity)

Mixed Morpholino Injected

Morpholino Injected

Rescue-p53

Uninjected

Primer zCdkn1a

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

acknowledgements
Acknowledgements

Rita Shiang

Sami

Omer

Balsam

Jim Lister

Brendon

Leyla