Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

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In order to gain a deeper insight into the genetic regulation of cytokine-determined and immunoglobulin/ T cell receptor based signaling in lymphocytes, efforts to use RNA interference (RNAi) technology as a screening tool have been undertaken. Selected libraries of shRNAs and siRNAs have been obtained. Introduction of siRNAs into resting CD4 T cells using Amaxa transfection technology has been achieved and, in test systems, impressive inhibition of expression and function of kinases such as Jak3 has been obtained. Currently, we have assembled a protein tyrosine kinase library and a protein tyrosine phosphatase library that will be tested for its capacity to inhibit or enhance the induction of Th2 phenotype by naive CD4 T cells. To this end, we have developed "recombineered" mice that faithfully expresses DS-Red as a surrogate for IL-13 and others that express Am-Cyan as a surrogate of IL-4 and destabilized DS-Red as a surrogate for IL-13. Thus, cells can be tested for induction or inhibition of surrogate expression and thus restimulation can be avoided. We anticipate both cloning cells that have been selected (i.e. either induced or suppressed, depending on the experimental protocol) and determining what shRNAs they have incorporated or using bulk induced or suppressed cells, carrying out microarray analysis of the shRNAs expressed by the bulk selected populations.