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Sandhya Varma S.
Reg No:1741110035
Under the guidance of
Dr. S. KRISHNAKUMAR
Department of Nanobiotechnology
Vision Research Foundation
Sankara Nethralaya
Chennai
INDUCTION OF APOPTOSIS USING TISSUE SPECIFIC PROMOTER GUIDED SUICIDE GENE TRANSFECTION IN NON INVASIVE RETINOBLASTOMA CELL LINE
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INTRODUCTION Retinoblastoma :Tumor arising from the
retina Suicide Gene Therapy: a promising
approach in cancer treatment CMV promoter : non specific delivery of the
gene of interest Viral vectors: safety concern, infects only
first few layers of the cells Novel approach to reduce toxicity and
improve cell survival Non viral vectors: Efficient and safe TSPs : specific for tumor cells
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Fig 1 : Picture showing unilatrral retinoblastoma (William F. Deegan III Children’s National Medical Center
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Fig 2: Pathology photograph showing the damage caused from retinastbloma. (William F. Deegan III Children’s National Medical Center)
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INTRODUCTION Apoptosis : redundant, damaged or infected
are elimination (Kerr et al.,1972) Cell death choreographers: caspases Disturbances : Degenerative disorders to
auto immunity and cancer (Cory and Adams 2002)
Inhibitors of apoptosis Over expression: Cells lose their ability to
undergo apoptosis
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OBJECTIVES1. Propagation of suicide gene plasmid2. To transfect the suicide gene plasmid
in non invasive Retinoblastoma cell line
3. To validate the transfection efficiency by various post transfection techniques
4. To study apoptotic effects induced by the suicide gene in the transfected non-invasive retinoblastoma cell line
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MATERIALS
Plasmid construct:
Cell line: Non-invasive retinoblastoma cell line
Vector: PUC18 Gene: suicide gene Promoter: TSP
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METHODOLOGY
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Detection of the suicide gene expression at mRNA level by real time PCR
Confirmation of the expression of the suicide protein by indirect Immunofluoresence
Detection of apoptosis by annexin V and PI staining
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RESULTS
Transformation of the suicide gene plamid in E.coli DH5α
Fig 3: LB +amp(100ug/ml) plate showing transformed colonies
OBJECTIVE 1: Propagation of suicide gene plasmid
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Plasmid mini preparation by alkaline lysis
S. No Sample Conc (ng/µl) 260/280 260/230
1 Suicide gene plasmid
4186.44 1.73 1.65
Fig 4: 1% agarose gel showing bands of the suicide gene plasmid (~10 kb)
Table 1: Quantification of the suicide gene plasmid
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Fig 5:Picture showing non invasive Retinoblastoma cells under phase contrast microscope (40X)
OBJECTIVE 2: Transfection of the suicide gene plasmid in non-invasive retinoblastoma cell line
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Isolation of RNA from the transfected non invasive retinoblastoma cell line
S.no Sample Conc(ng/ul) 260/280 260/230
1Total RNA from the transfected non-invasive retinoblastoma cell line
72.62 1.83 2.2
Table 1: Quantification of RNA using NanoSpec
Objective 3: To validate the transfection efficiency by various post transfection techniques
Fig 6: 1% agarose gel showing bands of the suicide gene plasmid (~10 kb)
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cDNA conversion of suicide gene mRNA by Reverse transcriptase PCR
S.No Sample Conc (ng/ul) 260/280 260/230
1 cDNA 82.49 1.78 1.86
Table 2: Quantification of the cDNA using NanoSpec
Amplification of the cDNA of suicide gene by PCR
Fig 7: 0.8% agarose gel showing the amplified product of the suicide gene(100 bp)
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Detection of the suicide gene expression at mRNA level by real time PCR
Suicide gene transfection in non invasive retinoblastoma cell line
Fig 8: Chart showing the over expression of suicide gene in transfectednon invasive Retinoblastoma cell line(~10 fold increase)
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Confirmation of expression of suicide gene protein in non- invasive retinoblastoma cell line by indirect
Immunofluoresence
Fig 9: A:Transfected (40X)(DAPI+FITC) B:Transfected(40X)(DAPI+FITC) under phase contrast C:Untransfected 40(DAPI+FITC). D:Untransfected (DAPI+FITC) under phase contrast.
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Apoptosis detection by Annexin V and PI staining
OBJECTIVE 4: Study of apoptotic effects induced by the suicide gene in the transfected retinoblastoma cell line
Fig 10: A: Graph showing the gated non invasive retinoblastoma cells transfected with suicide gene plasmid (10,000 cells) B: Quadrant graph with 61.84% of the transfected cells showing early apoptosis and 35.02% of the cells showing late apoptosis.
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STATUS REPORTS.NO OBJECTIVE STATUS
1 Propagation of suicide gene plasmid
Completed
2 To transfect the suicide gene plasmid in non invasive RB cell line
Completed
3 To validate the transfection efficiency by various post transfection techniques
Completed
4 To study apoptotic effects induced by the suicide gene in the transfected RB cell line
Completed
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REFERENCES William F. Deegan III Retinoblastoma: A Review of Current Treatment
Strategies Children’s National Medical Center Washington, D.C. Knudson, A. G. Mutation and cancer: Statistical study of
retinoblastoma. Proceedings of the National Academy of Sciences(1971) 68, 820–823
Kerr, J.F.R., Wyllie, A.H., and Currie, A.R.. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer (1972) 26: 239–257.
Cory, S. and Adams, J.M.. The Bcl2 family: Regulators of the cellular life-or-death switch. Nat. Rev. Cancer (2002) 2: 647–656.
Young JL, Smith MA, Roffers SD, Liff JM, Bunin GR. Retinoblastoma. In: Reis LAG, Smith MA, Gurney JG, et al, eds. Cancer incidence and survival among children and adolescents: (1999) United States SEER program 1975–1995. Bethesda, Md: National Institutes of Health.
Zeng B Zhu, Sharmila K Makhija, Baogen Lu, Minghui Wang, Lioudmila Kaliberova, Bin Liu, Angel A Rivera, Dirk M Nettelbeck, Parameshwar J Mahasreshti, Charles A Leath, III Shannon Barker, Masato Yamaoto,1 Fengzhi Li, Ronald D Alvarez, and David T Curiel. Transcriptional targeting of tumors with a novel tumor-specific survivin promoter. Cancer Gene Therapy: (2004)11; 256–262
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THANK YOU!!!