Generation of lentiviral-induced BMP reporter HEK293A

Abstract

BMP(bone morphogenic protein) is a signaling molecule which is known to play critical role in embryonic development and tissue homeostasis. Due to its importance in development, the expression level and pattern of BMP have been observed in vertebrate model systems and mammalian cell lines in various studies. To study the expression of BMP in model systems, it is important to make the expression level easy to measure and analyze by using reporter genes. Especially, using visually analyzable genes such as fluorescent proteins can be useful for analyzing the expression level in single cell level and therefore observing heterogeneity of expression levels among the cells. In this research, a vector construct with BRE(bmp response element)-mCherry sequence was generated for lentiviral transfection and transfected into HEK293a cells. Although the construct was succesfully generated, the mCherry fluorescence level of the BMP6-treated transfected cells were not significantly different from that of untreated cells. Judging from the RNA level and protein levels, the expression level of mCherry is consistently high due to high basal level of pSMAD1/5 in HEK cells. Therefore, it would be necessary to adjust the experimental conditions or use another cell line in order to troubleshoot this pSMAD level problem.