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Immortal bovine mammary epithelial cell lines are useful for providing an efficient indicator for transgene expression and for the technological improvement of genetic modification. The preparation of hTERT (human telomerase reverse transcriptase)-mediated immortalized MECs (mammary epithelial cells) requires a down-regulation of p16INK4a. Here, we report the establishment of two immortal bovine MEC lines by expression of hTERT gene alone under serum-containing culture conditions. This two cell lines maintain the general characteristics of MECs and have been stably passed more than 200 generations accompanying telomere extension, and were identified as non-malignant transformation. Investigation on transcriptional profile showed a similar down-regulation in both p16INK4a and p53. By comparing with non-immortal hTERT-positive MECs, we speculated that there are some spontaneous p16INK4a-reduced cells under normal culture conditions and the immortalization required for a co-ordinate repression of p53 and p16INK4a signalling pathways. Interestingly, two immortal cell lines showed a significant distinction in proliferation rate, implying that other mechanisms might be involved in proliferation control. [ABSTRACT FROM AUTHOR]