Many tumor suppressor genes are involved in the multistep process of neoplastic development. However, the signaling mechanism that underlies the development of tumors has not yet been completely elucidated. Therefore, Discovery of a novel tumor suppressor gene plays a crucial role in our understanding of the development and progression of malignant tumors. Chromosome engineering technique that base on Microcell-mediated chromosome transfer （MMCT），which can be generally used to the introduction of a single chromosome to a variety of tumor cells, is one of effective approach for mapping and identification of tumor suppressor genes. We have identified paired-like homeodomain 1（PITX1）gene as a novel telomerase negative regulatory factor that inhibit the expression of telomerase reverse transcriptase （TERT）using chromosome engineering technique. Here, we describe a unique strategy from mapping to identification of tumor suppressor gene by using MMCT approach.