PLASMID RECOMBINANT CONSTRUCTION OF SOX2 PROTEIN CODING OF PLURIPOTENCY FROM INDONESIAN BREAST CANCER STEM CEL L
Hanifah Rahmi1, Amarila Malik2, Septelia Inawati W3
1 Pharmacy, Faculty of Pharmacy and Science UHAMKA
2Microbiology and Biotechnology, Faculty of Pharmacy UI
3Biochemistry and Molecular Biology, Faculty of Medicine UI
GLOBOCAN 2012, International Agency for Research on Cancer
Introduction
Breast cancer is the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012 (second most common cancer overall). This represents about 12% of all new cancer cases and 25% of all cancers in women. It is the fifth most common cause of death from cancer in women.1
Cancer Therapy
Surgery
Chemotherapy
Hormon Therapy Radiation
Most therapies are directed at the fast
growing tumor mass but not the slow dividing cancer stem cells. It is often considered to be associated with chemo- resistance and radio- resistance that lead to the failure of traditional therapies.2
• CSC elimination is a new therapy strategic which high potencial to treat a cancer disease.
Introduction
•
Cancer stem cells (CSCs) are cancer cells that possess characteristics associated with normal stem cells.
•
CSCs have pluripotential properties by major markers such as Oct-4, Sox2, c-Myc, and Klf4.
•
Sox2 has an important role in cells determination, differentiation, and proliferation. Chen et al. (2008) discovered the correlation between the increase of Sox2 gene expression and the severity of breast cancer.
•
The particular characteristics of Sox2 could be used to estimate the presence of cancer stem cells in a group of cancer cells. Therefore, Sox2 can be used as a marker to determine the location of cancer stem cells in cancer cells.
Introduction
To construct SOX2 recombinant
plasmid that can be utilize to
develop breast cancer antibody
and as biomarker for cancer.
Research Methods
RNA isolation Human Breast CSC (CD 24-/CD44+)
SOX2 sequence identification Primer design
Reverse Transcriptase PCR
Electrophoresis and Gel Extraction
Insert SOX2 to pET101 vector
Metode Penelitian
pET101 vector with insert
Transform into TOP10 E.coli cells
Colony PCR
Sequencing
Human Breast CSC (CD24-/44+)
Breast CSC was
obtained from previous research which were sorted based on cell surface specific markers.
Design Primer
SOX2 primer (NM_003106.3) was designed from nucleotide base to 558-737
Forward primer
5'- CACCATGAACATGATGGAGACGGAGCT-3'.
Reverse primer
5'-GGGCCGGTATTTATAATCCGG-3'.
The length of Sox2 product generated from this primer pair
was 330 bp.
Reverse Transcriptase PCR analysis of SOX2
The RT-PCR product of Sox2 gene had a single band located as expected to the benchmark length of the Sox2 product, 330 bp.
Gene Transformation
Control
Competent cell colonies with SOX2 insertion In agar medium, 15 colonies with SOX2 insertion
was detected while in the control medium was unidentified colony.
Analysis PCR result of 15 colonies shows that all colonies contained SOX2 gene.
Colony PCR
To determine if the colonies contain Sox2 gene insertion.
Sequencing of PCR colony
Sequencing was conducted by 1st-Base with two reactions: forward and reverse. The alignment of nucleotide bases was arranged using Blast software on NCBI. BLASTN analysis results depicted 92% identity to SOX2 sequence with access code NM_003106.3
