Compton S T, Henning K A, Chen M, Mansoura M K, Ashlock M A
Vector Development Section, Genetics and Molecular Biology Branch, Building 49, Room 3A20, National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, MSC 4442, Bethesda, MD 20892, USA.
Nucleic Acids Res. 1999 Apr 1;27(7):1762-5. doi: 10.1093/nar/27.7.1762.
The transfer of high molecular weight (HMW) DNA into mammalian cells is an important strategy for assessing human gene expression and chromosome structure and function. However, using current methods, it is difficult to dependably prepare intact HMW DNA because of the susceptibility of the DNA to degradation and physical shearing. Here we describe a strategy whereby intact artificial chromosome DNA (as large as 1 Mb) can be routinely prepared from yeast. Strict adherence to this protocol has resulted in: (i) >90% of liquid DNA preparations containing largely intact DNA; (ii) transfection efficiencies for the development of stable human clonal cell lines ranging from 5 x 10(-7) to 8.8 x 10(-5); and (iii) the presence of markers from both YAC arms in 30-42% of the human fibrosarcoma cell HT1080 clones and 100% of the CF lung epithelial cell lines IB3-1 and CFT1 clones, suggesting that the HMW DNA is potentially intact in a substantial proportion of clones. Using this protocol for DNA preparation, successful transfection of functional 1 Mb human artificial chromosome DNA into human cells has also been achieved. This methodology should prove useful to those interested in using HMW human DNA for gene expression and functional analysis or for linear artificial chromosome construction, since integrity is absolutely critical for the success of these studies.
将高分子量（HMW）DNA导入哺乳动物细胞是评估人类基因表达、染色体结构与功能的一项重要策略。然而，使用当前方法，由于DNA易降解和物理剪切，难以可靠地制备完整的HMW DNA。在此，我们描述一种策略，通过该策略可常规地从酵母中制备完整的人工染色体DNA（长达1 Mb）。严格遵循此方案已带来以下结果：（i）>90%的液体DNA制剂含有大部分完整的DNA；（ii）稳定人类克隆细胞系的转染效率范围为5×10⁻⁷至8.8×10⁻⁵；（iii）在30% - 42%的人纤维肉瘤细胞HT1080克隆以及100%的CF肺上皮细胞系IB3 - 1和CFT1克隆中存在来自两个YAC臂的标记，这表明在相当比例的克隆中HMW DNA可能是完整的。使用此DNA制备方案，还成功地将功能性1 Mb人类人工染色体DNA转染到人类细胞中。该方法对于那些有兴趣使用HMW人类DNA进行基因表达和功能分析或用于线性人工染色体构建的人应是有用的，因为完整性对于这些研究的成功绝对至关重要。
