New reagents for improved in vitro and in vivo examination of TGF-β signalling.

Transforming growth factor-β (TGF-β) signalling controls many aspects of cell behaviour and is implicated as a key regulator in tumour formation and progression. However, evaluating levels of active TGF-β in culture medium or patient plasma and gaining definitive information regarding the activity of downstream substrates such as Sma- and Mad-related protein 3 (Smad3) in vivo with accuracy and sensitivity has been problematic. Therefore, to overcome these technical issues we have created a NIH3T3 cell line with stable pCAGA(12)-luc expression that can now be utilised to detect TGF-β activity with high sensitivity. In addition, we have created an adenoviral Smad3 luciferase reporter construct pAd.CAGA(12)-luc to successfully infect cells for in vitro assays, or prior to injection into mice and used to measure transcriptional activity in vivo. Thus, the NIH3T3-pCAGA(12)-luc cell line and the pAd.CAGA(12)-luc adenovirus will be extremely useful tools to measure TGF-β signalling activity with far greater efficiency and reliability compared to original and currently used reagents.