Cheung Wing, Kotzamanis George, Abdulrazzak Hassan, Goussard Sylvie, Kaname Tadashi, Kotsinas Athanassios, Gorgoulis Vassilis G, Grillot-Courvalin Catherine, Huxley Clare
Division of Natural Sciences, Imperial College London, London, UK.
Bioeng Bugs. 2012 Mar-Apr;3(2):86-92. doi: 10.4161/bbug.18621. Epub 2012 Mar 1.
Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.
将大型完整载体高效递送至哺乳动物细胞仍然存在问题。在此，我们评估通过细菌侵袭将两个大小超过150 kb的大型细菌人工染色体（BAC）递送至各种细胞的情况。首先，我们确定了几种药物对小质粒向不同细胞系进行细菌递送的影响。除了溶酶体亲和性药物氯喹外，大多数测试药物仅在某些细胞系中使总体递送效率略有提高，而氯喹在B16F10细胞中可使递送效率提高6倍。在将大型完整BAC递送至小鼠细胞方面，发现细菌侵袭比脂质体转染具有显著优势，可使100%的克隆含有完整DNA。此外，对存在于其中一个BAC中的人类次黄嘌呤磷酸核糖转移酶（HPRT）基因从其基因组位点进行表达评估，结果表明含HPRT的BAC在小鼠B16F10细胞中发生了单拷贝整合，并且每个人类拷贝的HPRT表达量是每个内源性小鼠拷贝的0.33倍。这些数据提供了新的证据，表明细菌递送是将大型完整治疗性基因转移至哺乳动物细胞的一种便捷且高效的方法。
