Montigny W J, Houchens C R, Illenye S, Gilbert J, Coonrod E, Chang Y C, Heintz N H
Department of Pathology, University of Vermont, Burlington, VT 05405, USA.
Nucleic Acids Res. 2001 May 1;29(9):1982-8. doi: 10.1093/nar/29.9.1982.
Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.
将大的DNA分子导入培养细胞存在困难，这阻碍了对完整哺乳动物基因和其他遗传区域的实验研究。此前我们已经表明，GST-Z2是一种蛋白质，它包含三个锌指结构以及来自多聚锌指蛋白RIP60的富含脯氨酸的多聚化结构域，并与谷胱甘肽S-转移酶（GST）融合，在体外介导DNA结合和环化。原子力显微镜显示，GST-Z2能够将130 - 150 kb的细菌人工染色体（BACs）浓缩成包含多个DNA环的蛋白质-DNA复合物。用阳离子脂质将DNA环浓缩到Z2蛋白-BAC DNA核心复合物上，形成的颗粒能够很容易地转移到多种培养细胞类型中。通过GST-Z2将含有扩增的二氢叶酸还原酶（DHFR）基因的总基因组线性DNA转移到DHFR(-)细胞中，其转化率比磷酸钙共沉淀法高10倍。用含有人类TP53基因位点的BAC转染中国仓鼠卵巢细胞后表达了p53，这表明在GST-Z2介导的基因转移后，天然启动子元件具有活性。由于GST-Z2介导的DNA浓缩不需要在DNA底物中引入特定的识别序列，RIP60的Z2结构域介导的浓缩可与多种其他试剂结合使用，为将大基因导入哺乳动物细胞提供一个灵活且高效的非病毒平台。
