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• We aimed to demonstrate its functionality by inserting it into pTRE-Luc vector.
• We expect that HRE, as an enhancer, would activate the promoter existing on the downstream region of it, depending on the level of HIF1alfa in the media which is increased in hypoxic conditions.

Expected To understand which colony our gene is inserted among the colonies that we transformated pHRE-luciferase vector, we expected the picture above when we perform PCR when we use pTRE-Luc forward and MCS reverse primers.

Experimented From the samples we perform colony PCR by using pTRE-Luc forward and MCS reverse primers, we obtained a band in 428 bp line. This image proves that our HRE sequnce is inserted into the vector, successfully.

Luciferase Assay

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NF-kappaB (NF-kB) proteins comprise a family of structurally-related eukaryotic transcription factors that are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. In some circumstances, NF-kB/IkB complex can be separated by external effects such as radiation, cellular stress, pathogens, inflammation etc. In this case, NF-kB can enter into nucleus and integrate with compatible kB-RE sites in order to initiate transcription.

We cloned kB-RE and inserted it into the downstream region of CMV mini promoter as it’s shown above.

NF-kappaB (NF-kB) was synthesized to GenScript™ company and it came in pUC57 plasmid. We digested it with BamHI & PstI and exposed it to Antarctic phosphatase.

Afterwards, we purified our part via phenol chloroform method. We performed the same procedures onto the pTRE-luciferase vector. Eventually, we ligated them.

We inserted our plasmid (pTRE-luciferase kB-RE) into DH5α strain and performed colony PCR by using CMV forward and kB reverse primers. At the end of this experiment, we expected a band seen in the 20-30 bp line.

And we observed correct bands in the expected region.

Reference

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ODD SYNTHESİS FROM HEP2G VİA PCR

Expected

Experimented

ODD (Oxygen Dependent Degredation) domain of HIF-1α was synthesized through liver cDNA using PCR with Sall enzyme restriction cites placed at the starting and ending points of the domain.

The PCR product was purified using the Phenol Chloroform method. Following the isolation, the ODD and pTet-Off vector were cut using the Sall restriction enzyme and then ligated. Thus, the ODD insert was placed in between the tetR(DNA binding domain) and VP16(Transactivating domain) of the pTet-Off vector.

COLONY PCR

Expected

Experimented

The DH-5α E.coli strains were transformated and, using CMV forward and SV40 polyA reverse primers, colony PCR was conducted and the vectors, in which the inserts were placed, were elected.

CUT-CHECK

Expected

Experimented

Since the ODD insert’s contained the same restriction cite on both ends, the colonies that entered the sequence from the right end were cut-checked using EcoRI and BamHI restriction enzymes and the colony containing the desired vector was selected.

ODD Results

ODD (oxygen dependent degradation) domain, which is present in the HIF-1α (hypoxia inducible factor) protein that it activates in lower oxygen levels and breaks down in intermediate oxygen levels, is a protein domain that plays a key regulatory role in the transcription of the HIF-1α factor. In intermediate oxygen levels, the ODD domain of the HIF-1α protein is hydroxilized by the hydroxilase enzyme and the hydroxilized HIF-1α enzyme breaks down through ubiquitin attachment. Thus, the ODD causes the regulation of a transcription factor, which is active in hypoxic conditions and inactive in normoxic conditions. The Tet-Off is a strong system composed of two strong plasmids. Of the two plasmids that form this system, the TetR-VP16 fusion protein produced by the first plasmid acts as a transcriptionary factor regulating the Tet operator sequence of the second plasmid(pTRE). (TetR: DNA Binding Domain, DBD; VP16: Transactivating domain, TAD).

The Tet-Off system can be inhibited using tetracyclane.

In this study, through placing the ODD region of the HIF-1α in between the synthetic TetR - VP16 transcription factors (which are not present in mammallian cells and have been used in molecular biology experiments for a long time), the transcription factor was designed to gain sensitivity to oxygen. The therapeutic genes in the pTRE genes can be synthesized as sensitive to the hypoxic conditions, controlled by the TetR-ODD-VP16 transcription factor.

Luciferase Assay

To control the functionality of the TetR-ODD-VP16 system, the HEK 293T and Hep G2 cell lines were cotransfected using the pTET Off-ODD and pTRE-Luc vectors.

100 µM of CoCl2 was added to the cell medium to establish 1% O2 in the medium. The cells were collected 9 hours later. Luminometric measurement under Thermo Varioscan for 613 nm was done and the data of the following graph were acquired:

The observation of the operational level of the TetR-ODD-VP16 did not yield fruitful results as the cells reached out of the dish when 100 µM of CoCl2 were added to HEK 293T medium.

When the Tet Off-ODD and pTRE-Luc vectors were cotranfected to Hep G2 cells in hypoxic medium, there was a 4 times increase in Luciferase concentration in respect to normoxic medium.

In the light of these results, it is possible to say that the Tet-ODD-VP16 system was successfully synthesized and a novel hypoxia inducible system was introduced for future use in studies indulged into examining hypoxic conditions.

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Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein involved in the breakdown of blood clots. It is a serine protease (EC 3.4.21.68) found in endothelial cells that can be secreted into the plasma as well as the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Due to its ability of working on the clotting system, tPA is used in clinical medicine to treat embolic or thrombotic stroke. However, use is contraindicated in hemorrhagic stroke and head trauma.

We questioned the best possible was of measuring the tPA enzyme activity and sought the answer to this question by examining the yield of a tPA catalyzed reaction. We searched the sector and discovered the Human tPA Activity Kit of the company ASSAYPRO which we then ordered to use. In the reaction which catalyzes tPA, our aim was to show that tPA was active when plasminogen was transformed to plasmine.

To acquire the tPA gene, the tPA forward and tPA revers primers were synthesized from the cDNA’s we were in possession of. Using these primers, we acquired the tPA genes by performing the PCR of the cDNA. The head and neck cancer cell line was used as the source for cDNA.

GENE SYNTHESIS FROM cDNA OF 64A CELL LINE

Expected The base length of tPA is 1762 bp’dir. The electrophoresis of the PCR was expected to show a base length of around 1700 bp. The image below shows the expected result of the electrophoresis.

Experimented Through the primers that we ordered and the cDNA, we acquired tPA inserts. The insert was, as expected, portraying that the base length was around 1700 bp.

CLONING CONTROL-1

Expected We used EcoRI and BamHI enzymes to cut the inserts and pTRE vectors which was followed by the ligation of our insert and vector using the ligation process. We then transformed our plasmids to The E. coli’nin DH5-α strains. Colony PCR was then conducted to verify the accuracy of our ligation.

Experimented The specified primers were put into colony PCR. As it can be seen in the results above, a right insert was not achieved. This process was repeated several times but no result was achieved. Seeing that ligation did not provide a solution to the problem, synthetically produced inserts were ordered.

The tPA gene which was ligated with the synthetically synthesized Puc57 vector was cut using the EcorI and BamHI restriction enzymes and then ligated with pTRE vector to be transformed into the DH5-α strain. We again used Colony PCR to control the accuracy of our ligation.

CLONING CONTROL-2

Expected The received genes were ligated with the pTRE vector, and then transformated to E.coli strands DH5-α. To test the validity of the transformation, we again applied colony PCR through CMV forward and SV40 reverse primers. The expected base length was 1984 bp.

Experimented Colony PCR was applied to the inserts acquired from transformation and ligation. As it can be seen in the figure above, the second colony contains the appropriate base length in respect to the ladder.

WESTERN BLOTTİNG

Expected The confirmed genes were transfected into HEK293 cells. The lysates acquired from the cells were run through Western Blot. tPA, that is known to have 63 kD, was expected to have the image on the left. http://www.emdmillipore.com/TR/en/product/Anti-tPA-%28Tissue-Plasminogen-Activator%29-Antibody%2C-clone-GMA-043,MM_NF-05-883

Experimented The bands in the Western Blot were accurate. The transfection of the inserts were verified and assays of tPA were prepared from the lysates.

After having proven the presence of tPA expression in the cells transfected with Western Blotting, the concentration of the amount of tPA in the cell and the amount of secreted tPA were measured using the ‘Human tPA Activity Kit’ to show that the expressed proteins are functionally active.

The real parameter of the measurement in the Assay was the product of the reaction of the plasmin enzyme. Since tPA shifts the inactive plasminogen to active plasmine, the measured value also presents tPA activity. The yield gives absorbance at 405 nm.

tPA ASSAY

Experimented

According to the tPA assay results we obtained, we were able to prove that the tPA enzyme could be produced and secreted from the cell successfully hence also showing that tPA is functionally active.

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