1. Induction of apoptosis using tissue specific promoter guided suicide gene
transfection in non-invasive retinoblastoma cell line
Sandhya Varma S.1 and S.Krishnakumar2
1Department of Genetic Engineering, SRM University, Chennai 6030203, India.
2Department of Nanobiotechnology, Vision Research Foundation, Sankara Nethralaya, Chennai
ABSTRACT
Suicide gene therapy is a promising
therapeutic approach for cancer. Targeted
expression of desired therapeutic proteins
within the tumor is the best approach to
reduce toxicity and improve survival. Recently,
viral vectors are substituted by non-viral
vectors and is proven to be an efficient means
of gene delivery. The most common promoter
used in gene therapy is CMV promoter.
Though CMV promoter has high efficiency, it
is universal promoter and results in the
delivery of the gene of interest in all tissues.
This drawback is countered with the help of
Tissue Specific Promoters. In this study, we
delivered a suicide gene using a tissue
specific promoter in retinoblastoma cell line to
induce apoptosis guided suicide gene in a
non-viral vector to study the efficiency of the
suicide gene in induction of apoptosis in non-
invasive retinoblastoma cell line.
INTRODUCTION
The main aim of gene therapy is the
development of efficient, non toxic gene
carriers that can encapsulate and deliver
foreign genetic materials into specific cell
types such as cancerous cells. During the
past two decades, enormous research in the
area of gene delivery has been conducted
worldwide, in particular for cancer gene
therapy application1. Viral vectors are
biological systems derived from naturally
evolved viruses capable of transferring their
genetic materials into host cells2. Although
non viral vectors are less efficient than viral
vectors3, they have the advantage of safety,
simplicity of preparation and high gene
encapsulation capability. Tumour specific
promoters (TSP) have emerged as highly
tumour specific and effective in selectively
allowing expression of the gene of interest in
cancer cells. The up regulation of TSPs in a
variety of tumours make it an ideal candidate
for tumour targeted gene therapy.
OBJECTIVE
1. To transfect the suicide gene plasmid in the
non-invasive retinoblastoma cell line
2. To validate the transfection efficiency by
various post transfection techniques.
3. To study the apoptotic effects induce by the
suicide gene in the transfected
retinoblastoma cell line
• Suicide gene plasmid isolation
• Transfection of non-invasive
retinoblastoma cell line with suicide gene
plasmid
• Detection of suicide gene expression
• Confirmation of protein expression by
immunofluorescence
• Apoptosis detection
METHODOLOGY
RESULTS
Fig 1: Graph showing the over expression of suicide gene in
transfected non-invasive retinoblastoma cell line (~10fold increase).
DISCUSSION
The purpose of this study was to generate a
non viral gene therapy vector with significant
and selective activity in tumour cells. We
developed tumour specific promoter driven
suicide gene whose expression in non
invasive cells lines were studied. More than
ten fold increase of the suicide gene
expression was confirmed. Protein level
expression was also studied. The suicide
gene plasmid induced apoptosis in more than
60% of non invasive retinoblastoma cells.
Hence, in future the suicide gene driven by
TSP in non viral vector can be used for the
treatment of retinoblastoma
REFERENCES
1. Scollay R (2001).Gene therapy: a brief
overview of the past,present, and future.
Ann. NY Acad. Sci. 953, 26–30
2. Cheung HH, Kelly N, Liston P, Korneluk R.
(2006) Involvement of caspase- 2 and
caspase-9 in endoplasmic reticulum
stress-induced apoptosis: a role for the
IAPs. Exp Cell Res 312: 2347−2357
3. Earnshaw WC. (1995) Nuclear changes in
apoptosis Current Opinions in Cell Biology
7:337:43.
Fig 8: A, B: pictures showing the stained nuclei (blue) and the suicide
gene protein (green) in non-invasive retinoblastoma cell line
transfected with suicide gene under fluorescent microscope(40X) C, D:
pictures showing the stained nuclei (blue) and suicide gene protein
(green) in untransfected non-invasive retinoblastoma cell line. The
suicide gene protein was present in transfected non-invasive
retinoblastoma cell line
Fig 9: 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
Fig 1: Figure showing the map of the suicide gene plasmid
Confirmation of protein expression by
immunofluorescence
Detection of suicide gene expression at mRNA
level
Apoptosis detection by annexin V and PI staining