RNA Silencing of Novel Gene Required For B Cell Development

B cell regulation is critical to the proper development of the immune system in mammals. Transcription factors and chromatin-remodeling events both occur during B cell development. Chromatin-remodeling events necessary for correct B cell differentiation are poorly characterized. Malfunction of B cell development has relevance to different health conditions, such as acute lymphoblastic leukaemia. The development of a novel mutant mouse strain, called Justy, provides a model for characterizing remodeling events necessary during pre-pro? Post? B cell development. Western blot analysis was done on multiple tissue samples to determine differential protein expression. Short hairpin RNA (shRNA) was developed to knockdown Justy protein expression and characterize the function and regulation of the Justy gene. Short hairpin (shRNA) designs were developed from the Justy sequence. The designs were derived via both Dharmacon and Sfold programming. Each 22 mer shRNA oligo thermostability profile was evaluated. The shRNA human miRNA30 (miR30) construct was chosen for endogenous recognition of shRNA. The pCR2.1 plasmid containing the construct was ligated into chosen retrovirus. Further experimental evidence of shRNA knockdown is needed via transfection and Western blot analysis