To begin, the authors constructed three different shRNAs that each targeted Trp53. These different shRNA constructs were then transfected into cultured cells and their effect on p53 production was assessed in vitro. Immunobloting revealed that cells transformed with the three shRNAs each reduced p53 expression to varying degrees. To determine whether the decreased p53 levels correlated to a phenotype, a colony-forming assay was used. Wild type cells tend to grow in a disperse fashion, while Trp53 knockout cells lose their contact inhibition and grow in distinct colonies. The shRNA that caused the greatest reduction of p53 protein levels also produced the most colonies in this assay, while the shRNA that only slightly reduced p53 protein levels produced the smallest number of colonies.
Once the shRNAs were characterized in vitro, the authors introduced them into transgenic mice and followed the progression of disease. All of the experimental mice showed symptoms associated with lymphoma development significantly earlier than control mice. Tumor progression, size and ultimately |
