2. EUKARYOTIC TRANSCRIPTIONAL
REGULATION
 Transcriptional regulation is said to be a control of gene expression
by the cell at transcriptional level.
 Transcription in the eukaryotic cells is regulated by the proteins which
bind to specific regulatory sequence and modulate the activity of
RNA polymerase.
 Sometimes, the packaging of the DNA into histones and modification
by methylation is involved into further complexity for gene regulation.

3. ONCOSTATIN-M
 Oncostatin-M is a cytokine.
 It belongs to interlukine-6(IL6) family
 It is molecular weight of 28 kd.
 Secreted by- Macrophages, T cells.
 Target cells- Tumor cells.
 Function- Inhibits the growth of the tumor cell.

4. PROMOTER OF ONCOSTATIN-M
IMPORTANT CORE ELEMENTS
GC-rich element.
STAT element.
CRE region.

5. The prime focus is to isolate the promoter region
1 kb upstream of ONCOSTATIN-M gene and clone it to
a expression vector for LARGE SCALE PLASMID
ISOLATION for further assay.
AIM OF THIS PROJECT

6. WORK FLOW

7. Culture U937 cell line
Isolation of genomic DNA
Amplification of full length promoter region of osm gene from the genomic DNA
WORK DONE PREVIOUSLY

8. RESULT- FULL LENGTH PROMOTER REGION(PON12)
AMPLIFICATIO FROM GENOMIC DNA
960 bp
1 2 3 4 5
Lane 1-1000 bp ladder
Lane 3-5-Amplified PON12
region

9. Ligation of the promoter region into TA cloning vector
Transformation into E.coli XL Blue cell
Colony selection and re-streaking for storage
Plasmid( pt-Pon12) isolation

10. RESULT-PLASMID ISOLATION OF pT-PON-12
1 2 3 4 5
Lane 1-PT-PON12 plasmid
Lane 3-Ptz plasmid without insert
Lane 5-PT-PON12 plasmid

11. Sub-cloning into pEGFP-1 vector with the help of directional
cloning by 2 different restriction enzymes (Eco RI and Bam HI)
Eco RI
Bam HI
Eco RI Bam HI
Pon12

12. RESULT-Restriction digestion of pEGFP-1 and pT-Pon12 plasmids
1 2 3 4 5 6 7
960 bp
Lane 1-1000 bp ladder
Lane 3-Uncut PEGFP-1
Lane 4-Digested PEGFP-1 by Eco RI, Bam HI
Lane 6-PT-PON12 digested by Eco RI , Bam HI
Lane7-PT-PON12 digested by Eco RI, Bam HI

13. Ligation and Transformation into E. coli XL Blue cells
Colony selection and re-streaking
Perform colony PCR for confirmation of clone
Selection of the positive colony

14. RESULT-Colony PCR from colony containing p-EGFP1-Pon12 plasmid with
specific primer Pon1 and Pon2
1 2 3 4 5 6 7 8
960 bp
LANE 1,3,4,8-Colonies picked from transformed plate
LANE 5- 1000 bp ladder

15. Inoculate this colony into LB +Amp media for large scale plasmid
culture
Grown overnight
Isolation of pEGFP1-Pon-12 plasmid by PEG and LiCl2 method
The isolated plasmid is used for the further assay

16. 1 2
LANE1-pEGFP plasmid without insert
LANE2-pEGFP plasmid with insert
RESULT- ISOLATION OF pEGFP1-Pon12 PLASMID

17. Thus we have successfully cloned and isolated PEGFP1 vector containing full
length osm promoter region .This clone can be further used for transfection and
subsequent reporter assay in mammalian system. This will help us study the role
of osm promoter region in its efficient transcription.

18. Dr SUMITA SENGUPTA
Department of Molecular Biology, Biophysics  Bioinformatics
UNIVERSITY OF CALCUTTA
All my guides, specially,
SRIMOYEE MUKHERJEE
PROSENJIT DAS
Head of The Department, Microbiology
Vijaygarh Jyotish Ray College
All my respectable professors, department of Microbiology
Vijaygarh Jyotish Ray College
CALCUTTA UNIVERSITY CENTRAL INSTRUMENTAL FACILITY