Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Mutagenesis in Ataxia Telangiectasia
Induced Pluripotent Stem Cells
Lucy Lin
Dr. Ronald P. Hart
Department of Cell Biology...
Ataxia Telangiectasia
• Ataxia = loss of motor control; Telangiectasia = spider veins
• Neurodegenerative, inherited disea...
ATM Kinase Protein
• 370 kilodalton
• Serine/threonine protein
• Member of the phosphatidylinositol 3-kinase related kinas...
DNA Damage Repair Cascade
Matsuoka et al., 2007
Induced Pluripotent Stem Cells
• Cells that can be reprogrammed from adult
somatic cells through transcription factors to
...
A-T Cell Lines are Pluripotent
Oct-4 Tra-1-60
Q1-SA
Q3-SA
Q3-SC
CAR3-SB
ATM -/-
ATM -/-
ATM -/-
ATM +/-
• CAR3 Cells (carrier): Deletion causing
frameshift on one allele and one normal
allele
• Q1 and Q3 Cells (A-T): Compound
...
Q1 and Q3 Allele Mutations
Hart, R. P., unpublished
Subject
Code
Age Sex Diagnosis Mutations iPSC Lines
JHU_Q1 23 F A-T c....
Actin
pChk2
Q3-SC Cells Induce pChk2
High levels of γH2A.X seen in Q3-SC cells
after X-irradiation
ATM +/- ATM -/- ATM -/- ATM -/-
Q3-SA and Q3-SC Cells are from the
same subject
Hoffman, G., unpublished
Hypothesis
• Spontaneous mutagenesis is exacerbated
by the absence of ATM-guided DNA
repair, which can lead to reversion o...
Methods
• Introduce DNA damage to the cells
through X-Irradiation to induce
mutagenesis and perhaps
reversion
• Incubate c...
pATM formation after irradiation seen in
all ATM -/- cell lines
Images and Data Collected by Gary Hoffman, 2015
Conclusions
• Further support of reversion in Q3-SC cells
• Possibility of reversion in Q3-SA cells
• Reversion in Q1-SA c...
Future Directions
• Repeat mutagenesis experiment with the
addition of staining for γH2A.X along with
pATM
• Establish IC5...
Acknowledgements
Hart Lab
• Ronald P. Hart, PhD
• Alana Toro-Ramos
• Gary Hoffman
• Michael Lazaropoulos
• Eileen Oni
• Ma...
References
1. Adams, B.R., Golding, S.E., Rao, R.R., Valerie, K. (2010). Dynamic dependence on ATR and ATM for double-stra...
Upcoming SlideShare
Loading in …5
×

SURF 2015 Mutagenesis in Ataxia Telangiectasia Induced Pluripotent Stem Cells

1,664 views

Published on

  • Be the first to comment

SURF 2015 Mutagenesis in Ataxia Telangiectasia Induced Pluripotent Stem Cells

  1. 1. Mutagenesis in Ataxia Telangiectasia Induced Pluripotent Stem Cells Lucy Lin Dr. Ronald P. Hart Department of Cell Biology and Neuroscience Rutgers, The State University of New Jersey Summer Undergraduate Research Fellowship 2015
  2. 2. Ataxia Telangiectasia • Ataxia = loss of motor control; Telangiectasia = spider veins • Neurodegenerative, inherited disease caused by mutation in ATM gene – Causes tumor formation – Impairs cerebellum – Prevents repair of broken DNA • Some symptoms – Nystagmus (rapid involuntary eye movement) – Ocular telangiectasia
  3. 3. ATM Kinase Protein • 370 kilodalton • Serine/threonine protein • Member of the phosphatidylinositol 3-kinase related kinase (PIKK) family  important for DNA repair regulation • Regulates DNA damage response cascade to arrest cell cycle for DNA repair Llorca et al., 2003
  4. 4. DNA Damage Repair Cascade Matsuoka et al., 2007
  5. 5. Induced Pluripotent Stem Cells • Cells that can be reprogrammed from adult somatic cells through transcription factors to become pluripotent Bellin, M., Marchetto, M. C., Gage, F. H., and Mummery, C. L., 2013 Retrovial infection with 4 transcription factors: Oct3/4, Sox2, cMyc, Klf4
  6. 6. A-T Cell Lines are Pluripotent Oct-4 Tra-1-60 Q1-SA Q3-SA Q3-SC CAR3-SB ATM -/- ATM -/- ATM -/- ATM +/-
  7. 7. • CAR3 Cells (carrier): Deletion causing frameshift on one allele and one normal allele • Q1 and Q3 Cells (A-T): Compound heterozygote - deletion causing frameshift on one allele and point mutation on other Hart, R. P., unpublished A-T Cell Line ATM Allele Mutations Lazaropoulos, M., unpublished Subject Code Age Sex Diagnosis Mutations iPSC Lines JHU_Q1 23 F A-T c.[1564delGA]; [7181C>T] Q1-SA JHU_Q3 8 M A-T c.[7792C>T]; [217_218delGA] Q3-SA Q3-SC JHU_CAR3 42 F Carrier Not tested CAR3-SB
  8. 8. Q1 and Q3 Allele Mutations Hart, R. P., unpublished Subject Code Age Sex Diagnosis Mutations iPSC Lines JHU_Q1 23 F A-T c.[1564delGA]; [7181C>T] Q1-SA JHU_Q3 8 M A-T c.[7792C>T]; [217_218delGA] Q3-SA Q3-SC
  9. 9. Actin pChk2 Q3-SC Cells Induce pChk2
  10. 10. High levels of γH2A.X seen in Q3-SC cells after X-irradiation ATM +/- ATM -/- ATM -/- ATM -/-
  11. 11. Q3-SA and Q3-SC Cells are from the same subject Hoffman, G., unpublished
  12. 12. Hypothesis • Spontaneous mutagenesis is exacerbated by the absence of ATM-guided DNA repair, which can lead to reversion of the ATM gene to produce functional ATM kinase –All ATM -/- cells are more sensitive to reversion than other ATM genotypes
  13. 13. Methods • Introduce DNA damage to the cells through X-Irradiation to induce mutagenesis and perhaps reversion • Incubate cells for another week to allow time for reversion • One week after the first irradiation, irradiate cells again to elicit pATM formation • After fixing and staining cells, images were taken using IN CELL Analyzer 6000, an automated confocal microscope X-Irradiation Double Strand Breaks
  14. 14. pATM formation after irradiation seen in all ATM -/- cell lines Images and Data Collected by Gary Hoffman, 2015
  15. 15. Conclusions • Further support of reversion in Q3-SC cells • Possibility of reversion in Q3-SA cells • Reversion in Q1-SA cells? Hart, R. P., unpublished S1981
  16. 16. Future Directions • Repeat mutagenesis experiment with the addition of staining for γH2A.X along with pATM • Establish IC50 of ATM inhibitor for control cells and genomically reverted A-T cell line Q3-SC
  17. 17. Acknowledgements Hart Lab • Ronald P. Hart, PhD • Alana Toro-Ramos • Gary Hoffman • Michael Lazaropoulos • Eileen Oni • Mavis Swerdel • Jennifer Moore, PhD • Angela Tiethof • Kunal Garg • Sri Puli Lab Collaboration • Lourdes Serrano, PhD • Zhiping Pang, PhD SURF Directors • Lauren Aleksunes, PharmD, PhD • Debra L. Laskin, PhD Funding • National Institutes of Health R25ES020721 Grant • A-T Children’s Project of the ATCP Foundation
  18. 18. References 1. Adams, B.R., Golding, S.E., Rao, R.R., Valerie, K. (2010). Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants. PLoS ONE 5, e10001. 2. “Ataxia Telangiectasia.” National Cancer Institute. National Institutes of Health, 26 Jan. 2006. Web. 25 July 2014. 3. Barzilai, A., Rotman, G., Shiloh, Y. (2002). ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage. DNA Repair (Amst) 1, 3-25. 4. Bellin, M., Marchetto, M. C., Gage, F. H., and Mummery, C. L. (2013). Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Biol 13, 713-726. 5. Biton, S., Barzilai, A., and Shiloh, Y. (2008). The neurological phenotype of ataxia-telangiectasia: solving a persistent puzzle. DNA Repair (Amst) 7, 1028-1038. 6. Kulkarni, A., Das, K.C. (2008). Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation. Am J Physiol Lung Cell Mol Physiol 294, L998-L1006. 7. Llorca, O., Rivera-Calzada, A., Grantham, J., and Willison, K.R. Electron microscopy and 3D reconstructions reveal that human ATM kinase uses an arm-like domain to clamp around double-stranded DNA (2003). Oncogene 22, 3867–3874. 8. Matsuoka, S., Ballif, B.A., Smogorzewska, A., McDonald, E.R. 3rd, Hurov, K.E., Luo, J., Bakalarski, C.E., Zhao, Z., Solimini, N., Lerenthal, Y., Shiloh, Y., Gygi, S.P., Elledge, S.J. (2007). ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160-1166. 9. Pang, Z.P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D.R., Yang, T.Q., Citri, A., Sebastiano, V., Marro, S., Südhof, T.C., Wernig, M. (2011). Induction of human neuronal cells by defined transcription factors. Nature 476, 220-224. 10.Sedgwick, R.P., and Boder, E. (1960). Progressive ataxia in childhood with particular reference to ataxia- telangiectasia. Neurology 10, 705-715. 11.Serrano, L., Liang, L., Chang, Y., Deng, L., Maulion, C., Nguyen, S., and Tischfield, J.A. (2011). Homologous recombination conserves DNA sequence integrity throughout the cell cycle in embryonic stem cells. Stem Cells Dev. 20, 363-374. 12.Shiloh, Y., and Ziv, Y. (2013). The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14, 197-210.

×