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

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

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)

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

 

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

 

 

MATERIALS

Plasmid construct:

Cell line: Non-invasive retinoblastoma cell line

Vector: PUC18 Gene: suicide gene Promoter: TSP

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

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

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

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

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)

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)

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)

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.

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.

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

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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