Treatment of epithelial 293T cells with thymocyte extract to induce RAG gene expression

Abstract

This thesis reports an in vitro reprogramming approach with pig thymocyte extracts to induce expression of two lymphoid-specific recombination activating enzyme genes, RAG1 and RAG2, in human kidney epithelial 293T cells. RAG2 was upregulated in extract-treated cells 8 days after extract treatment, suggesting that some functional changes may have taken place in 293T cells exposed to thymocyte extract. RAG2 upregulation was detected in high (22mg/ml) protein concentration thymocyte extract-treated cells. However, induced expression of RAG2 was transient and decreased to basic level within 23 days. This transient expression might be explained by the nature of RAG expression in vivo, or it might indicate, most likely, incomplete reprogramming. Furthermore, upregulation of RAG2 was not accompanied by RAG1 expression or changes in morphology and growth pattern of 293T cells. Thus, we conclude that although RAG2 upregulation is observed in extract-treated cells, further work is required to establish a proof-of-concept that RAG enzyme-initiated T cell receptor gene rearrangement can be manipulated experimentally in non-lymphocytic cells.