| Abstract [eng] |
P53 is an extremely important for cancer supression transcription factor. Thus, it would be beneficial to assess its transcriptional activity in living cells in real time. This can be achieved by creating its fluorescent reporter. The sensitivity of such a reporter to changes in transcriptional activity could be enhanced by destabilizing the fluorescent protein. The hygromycin resistance gene of the lentiviral "TET ON" system vector was replaced with the puromycin resistance gene. The PEST sequence of the mouse ODC1 gene was cloned into the pEGFP-C2 vector. The pEGFP-C2-PEST construct was sequenced. Then, both standard and destabilized eGFP variants were subcloned into the "TET ON" system vector. Corresponding cell lines were derived, and eGFP and deGFP expressions were induced in them. The comparison showed a dramatic difference between the intensities of eGFP and deGFP fluorescence. It was concluded that the PEST sequence is active. Then, the P21 promoter sequence with two P53 responsive elements was cloned into the pJET plasmid. The construct was sequenced. Then, the P21 promoter was subcloned into lentiviral plasmids before the sequences of eGFP and deGFP genes. For the transduction and validation of the obtained constructs, HCT116 and HCT116-p53-/- cell lines were selected. The derived lines were then exposed to fixed concentrations of P53 inducers - nutlin and oxaliplatin. It was found that the reporters respond to the existence of P53 and the induction of its transcriptional activity. Nutlin and oxaliplatin dose titrations were performed. Regression analysis showed that the system variant with the standard eGFP version is better for use with flow cytometry. Using the eGFP reporter version, the positive P53 status of the HepG2 cell line was confirmed. The sequencing of the coding region of the TP53 gene of PC1 and PC2 cell lines was performed. A new TP53 mutation c.1025delG (p.Arg342Glnfs*3) was identified. Using the created reporter, it was shown that P53 with this mutation has no transcriptional activity. Attempts were made to increase the specificity of the P53 reporter by replacing the P21 promoter with single and triple P53 response elements and different combinations of YB_TATA and minCMVmod minimal promoters. Combinations with the minimal YB_TATA promoter were inactive. Combinations with the minCMVmod promoter were only partially active, but specificity was increased. In conclusion, it can be stated that the P53 fluorescent reporter was created and successfully applied to determine HepG2, PC1, and PC2 cell lines‘ P53 transcriptional activity. However, its modification was unsuccessful. |
