Potter P M, Harris L C, Remack J S, Edwards C C, Brent T P
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Cancer Res. 1993 Apr 15;53(8):1731-4.
A synthetic oligonucleotide containing ribozyme sequences targeted to the 5' region of the human O6-methylguanine-DNA methyltransferase (MGMT) mRNA has been constructed. This ribozyme demonstrates cleavage activity in vitro in the presence of Mg2+. To determine whether this ribozyme can function in vivo, HeLa CCL2 cells were transfected with a mammalian expression vector containing the ribozyme sequence. Following selection and expansion of individual transfectants, a stable clone was isolated that lacks both MGMT mRNA and protein. Molecular analysis of this cell line indicates that in vivo cleavage of MGMT mRNA is responsible for the lack of MGMT activity.
构建了一种合成寡核苷酸，其含有靶向人O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）mRNA 5'区域的核酶序列。这种核酶在Mg2+存在的情况下在体外表现出切割活性。为了确定这种核酶在体内是否能发挥作用，用含有该核酶序列的哺乳动物表达载体转染了HeLa CCL2细胞。在对单个转染子进行选择和扩增后，分离出一个稳定的克隆，该克隆既缺乏MGMT mRNA也缺乏MGMT蛋白。对该细胞系的分子分析表明，MGMT mRNA在体内的切割导致了MGMT活性的缺失。
