Construct A Proliferative Specific Promoter For Gene Therapy Of Glioma On The Basis Of PCNA Promoter

Objective:In order to construct an ideal proliferative specific promter for gene therapy of glioma, we selected the wild type PCNA promoter as the prototype and observed the transcriptive activity and proliferative specificity of its different fragments in a glioma cell line TJ905and a nontumorous cell line293both qualitatively and quantitatively. On the basis of the above results, we choosed the one with the highest transcriptive acitivty and integrated proliferative specificity and introduced certain modifications to construct the recombinant promoter PTAI, thus to further increase the transcriptive activity and the stability of the transcriptional initiation site.Methods:1. The PCNA promoters of different fragments of-778bp～+122bp,-538～+62,-256～+62were amplified with polymerase chain reaction(PCR) method, utilizing DNA of human genome.2. After DNA sequencing with correct result,the fragments were inserted into the green fluorescent protein(GFP) report gene vector (pEGFP-1). Then transiently transfected into glioma cell line TJ905and immortalizated nontumorous cell line293,qualitatively observed the expression of GFP.3. To insert the different fragments into luciferase reporter vectors (pGL3-Basic pGL3-Ehance).Quantitatively observed the different fragments transcriptional activity by Dual-GloTM fluorescein test kit.4.To reconstruct the fragment of900bp named the promoter PTAI.After the sequencing with correct result,transiently transfected into TJ905and293cell lines.Qualitative and quantitative observed and then contrast with other fragments in order to determine the transcriptional activity and proliferating specificity of the vector.Results:1. Electrophoresis demonstrated that cloned PCNA promoters were correct and DNA sequencing showed the same sequences as registered in GenBank.2. Qualitative and quantitative observation showed all the fragments could mediate the report gene expression. But different fragments had different activity.Transcriptional activity of900bp was obviously higher than600bp and300bp in the TJ905and293cell line.In the TJ905cell the transcriptional activity of900bp was higher than in293cell.3.it was confirmed that the reconstructive PTAI was succeed by restricted enzyme analysis and DNA sequencing.4. through the test, transcriptional activity of PTAI was higher than900bp,especially inserted into SV40.but the activity of them had no statistics difference.Conclusions:The difference fragments of cloning and reconstruction have strong activity.especially when inserted SV40,the activity rasied5-8fold and has no influence in the tumor specificity. So it is possible to become a better promoter to use in the gene therapy.