Isolation of HPRT gene depleted human TK6 lymphoblastoid cell line through gamma irradiation

Ionizing radiation induced DNA DSBs can cause large deletions in the human genome. This property may be utilized for gene disruption to create knockout mutants. The X-linked hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene is commonly used as a reporter to evaluate the frequency and molecular nature of somatic mutations in the human population. Mutations at the HPRT locus are measured through a T lymphocyte cloning assay. This assay relies on lymphoblast feeder cells that support T lymphocyte proliferation via cell-to-cell contact and by helping in the expression of functional interleukin-2 receptors. However, the feeder cells must be HPRT-depleted to prevent feeder cell DNA from serving as substrate during molecular analysis of T lymphocyte mutations. To the best of our knowledge, the human lymphoblastoid TK6 cell line available in Indian cell repositories has an intact HPRT gene (HPRT+). In this study, the parent TK6 cell line was subjected to the clastogenic effects of a high dose gamma radiation to isolate TK6 cells with a total deletion of the HPRT locus. The HPRT-depleted cell line (TK6_LLrrL) was characterized by mPCR for the loss of exons and the extent of the deletion by flanking STS markers analysis. Further, the feeder cell function of TK6_LLrrL was evaluated by estimating the cloning efficiency of human peripheral blood lymphocytes. Thus, the study demonstrates the potential of low LET gamma radiation to generate gene knockout cell line. The method described here can be applied to various cell types to produce functionally null cell lines for gene-specific studies.