Title: Analysis of TNFAIP1 DNA-mediated Signaling Pathway and Promoter Activity in Human THP-1 Cells in Response to P.gingivalis/LPS
Bushra Aljahdali, Boston University
Feng Cao (Presenter)
Abdullah Bamashmous, boston university
Mansour Alasiri, Boston University
Serge Dibart, Boston University
Erdjan Salih, Boston University
Xiaoren Tang, Boston University
Objectives: TNFAIP1 is known to be induced by TNF-a. Previous studies report that increased expression of TNFAIP1 is involved in many inflammatory disorders. Our previous data showed that PI3K/AP-1 regulated TNFAIP1 gene expression and consequently TNF-alpha production in response to LPS/P.gingivalis. We are planning to confirm this mechanism in human THP-1 cells. However, we found that there is a very low transfection efficiency (≤2%) of TNFAIP1 cDNA in THP-1 because this cells are suspension. We therefore wanted to solve these problems above.
Methods: 1. TNFAIP1 cDNA construction
2. ELISA assay.
3. Assay for TNFAIP1 promotor activity.
Results: To raise its transfection efficiency, we therefore tried several methods such as Lipofectamine or electroporation. Finally, we discovered a simple but powerful method that helps us to raise the DNA transfection efficiency up to 10% in THP-1 cells without PMA treatment. This method has been proved and also can be used for DNA transfection in other suspension cells such as human Myeloma cells. We also found the sequence of TNFAIP1 promoter DNA and cloned it intopGL3-basic plasmid DNA. Finally, we further analyzed the TNFAIP1 promoter activity.
Conclusions: The method that we found in this application is simple and powerful for DNA transfection in the suspension cells such as THP-1.
TNFAIP1 promoter activity is regulated by some transcriptional factors.