The myriad of drugs, growth factors, scaffolds available for tissue engineering makes conventional methods for analysis of differentiation (RT-PCR, histology) impractical. To facilitate the online monitoring of proceeding chondrogenic differentiation in living cells we are developing lentiviral reporter plasmids. Thereby the expression of a fluorescent protein (e.g. Green fluorescent protein (GFP)) is driven by a chondrogenesis promoter (e.g. Collagen 2 and Aggrecan). Our online chondrogenic monitoring constructs are further improved by the integration of a second easily detectable marker protein (histone H2B). The constitutive expression of the second marker protein enables evaluation of transfection efficiency.
Besides their potential in online monitoring applications, tissue-specific promoters can be used in studies of cell state essential genes. To investigate the effect of gene silencing above-mentioned promoters drive the expression of small hairpin RNAs (shRNA). Similarly, these specific promoters could facilitate cell state specific overexpression of genes of interest.
Lentiviruses can transduce almost any mammalian cell, including actively proliferating and non-dividing cells, stem cells as well as primary cells. The reporter gene is integrated into the host cell. This enables stable and long-term reporter gene expression.