Larocca D, Kassner P D, Witte A, Ladner R C, Pierce G F, Baird A
Selective Genetics Inc., San Diego, California 92121, USA.
FASEB J. 1999 Apr;13(6):727-34. doi: 10.1096/fasebj.13.6.727.
We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.
我们已对丝状噬菌体进行基因改造，使其能够将基因传递给哺乳动物细胞。在先前的研究中，我们发现非共价连接的成纤维细胞生长因子（FGF2）可将噬菌体靶向COS-1细胞，从而实现报告基因的受体介导转导。因此，通常对哺乳动物细胞缺乏嗜性的噬菌体可适用于哺乳动物细胞基因转移。为了确定使用噬菌体介导的基因转移作为一种新型展示噬菌体筛选策略的潜力，我们用经过工程改造的噬菌体转染COS-1细胞，这些噬菌体被设计成在其表面衣壳上展示FGF2，并与次要衣壳蛋白pIII融合。免疫印迹和ELISA分析证实噬菌体衣壳上存在FGF2。与非靶向亲本噬菌体相比，靶向FGF2的噬菌体在COS-1细胞中获得了显著的转导。在过量游离FGF2存在的情况下，转导成功受到抑制，证明了其特异性。在证明了展示已知基因靶向配体的噬菌体能够实现哺乳动物细胞转导后，现在将噬菌体介导的转导应用于发现新型配体的筛选是可行的。
