Federation of American Scientists Case Studies in Dual Use Biological Research Module 5.0: Antibiotic Resistance Case Study
Topic: Experiments in Antibiotic Resistance

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
mouse survival time were once again proportional to the level of p53 protein knockdown associated with each shRNA. Based on these results it was concluded that shRNA is able to induce gene knockdown in mice. This demonstrated scaleable genetic control of Trp53 expression and, by extension, disease severity in a mammalian system.

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