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Optimizing polymerase chain reaction conditions for the human perlecan (HSPG2) promoter
Author(s) -
Richards Matthew Thomas,
Rohe Benjamin,
FarachCarson Mary C
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.524.12
Subject(s) - perlecan , stromal cell , promoter , biology , reporter gene , gene , microbiology and biotechnology , computational biology , cancer research , proteoglycan , genetics , extracellular matrix , gene expression
Perlecan, also called HSPG2, is a heparan sulfate proteoglycan predominantly located in basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells. The reactive stroma surrounding prostate cancer cell lines produces high levels of the protein which may play a role in delivery of growth and angiogenic factors, aiding survival and growth of metastatic tumors. The overall goal of this project was to study the promoter in order to understand the up‐regulation of perlecan in the tumor reactive stroma which occurs via transcriptional increases in perlecan biosynthesis. The sequence for the human HSPG2 promoter was found using public databases and compared to a published human sequence (Iozzo et al., 1997) and a mouse promoter sequence found in online databases. Several transcription factor binding sites of interest were identified for further study including NFkB, CREB, Smad3, Elk‐1, c‐Jun and TCF/LEF‐1. Currently we are working to build a promoter‐reporter construct by isolating the promoter region using polymerase chain reaction (PCR) amplification. Our current strategy seeks to optimize the PCR conditions with different sets of primers. The next step will be creating a fluorescence protein reporter construct of the full promoter sequence in order to test the effects of the identified pathways. This research was funded by the HHMI program and NIH/NCI P01 CA098912.