Objective: To construct pMCLacⅠ/Neo plasmid as an effective vector for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. Methods and Results: A pMCLacⅠ/Neo plasmid, containing 2 copies of lacⅠ gene as the mutation target genes, was designed and constructed by common molecular cloning techniques. In this plasmid, only one of the lacⅠ genes was under the control of CMV promoter and could be transcribed in human and mammalian cells. After structure identification by restriction analysis, pMCLacⅠ/Neo plasmid was further studied with its function. It was first introduced into E.coli cells to test the function of the 2 lacⅠ target genes. The results indicated that both lacⅠ genes could regulate the expression of lacZα reporter gene in DH5α host cells. Whether the 2 target genes could imitate the functional states of expressed and non-expressed genes in mammalian cells was also analyzed. A NIH3T3 cell line containing copies of a stably integrated pMCLacⅠ/Neo plasmid was established through liposome-mediated transfection, and the functional states of the 2 lacⅠ target genes in the cell line were analyzed with RT-PCR. The results showed that only one of the 2 lacⅠ genes could be transcribed in the NIH3T3 cells. Conclusion: A new plasmid as a vector for studying gene mutation, in which 2 lacⅠ target genes could imitate the functional states of expressed and non-expressed genes in human and mammalian cells respectively, is constructed.