Matthews K E, Mills G B, Horsfall W, Hack N, Skorecki K, Keating A
Toronto Hospital, Ontario, Canada.
Exp Hematol. 1993 May;21(5):697-702.
We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.
我们报道了一种简单、快速、高效且经济有效的将基因导入骨髓基质细胞和其他贴壁哺乳动物细胞的方法。我们的方法包括将细胞与玻璃珠在含有待转移DNA的溶液中短暂孵育。我们使用COS细胞（源自非洲绿猴的SV40转化肾细胞系）和氯霉素乙酰转移酶（CAT）的瞬时表达测定法对该技术进行了优化。影响基因转移的因素包括珠子的大小和状态以及DNA浓度，但不包括DNA构象。在β-半乳糖苷酶活性的瞬时表达测定中评估的基因转移效率，在未转化的人骨髓基质细胞和COS细胞中分别为5%和3%。在克隆形成的COS细胞中，使用选择标记新霉素磷酸转移酶证明了长期稳定表达，频率为27%。对抗性克隆的Southern分析表明，转移的DNA以低拷贝数整合到宿主细胞基因组的一个或两个位点。与电穿孔和DEAE-葡聚糖的比较表明，珠子转染比后者更有效，且比这两种方法中的任何一种成本更低。鉴于其简单性以及可以避免使用逆转录病毒序列，珠子转染可能是基因治疗中一种有吸引力的基因插入手段。
