Alexis Frank, Zeng Jieming, Shu Wang
Institute of Bioengineering and Nanotechnology, Singapore 138669.
CSH Protoc. 2006 Jun 1;2006(1):pdb.prot4451. doi: 10.1101/pdb.prot4451.
INTRODUCTION This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection using these complexes. The conjugation step involves the use of the succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional cross-linker, to form a stable bond between PEI and peptides containing thiol groups.
引言 本方案描述了用于靶向基因递送的聚乙烯亚胺(PEI)/DNA纳米颗粒的制备。这种递送策略通过增强基因载体进入所需细胞的能力并减少非靶细胞的摄取来提高基因转移效率。我们在此描述将靶向肽与PEI偶联的方法、使用偶联的PEI或未偶联的PEI与靶向肽形成DNA复合物的方法,以及使用这些复合物进行细胞转染的方法。偶联步骤涉及使用N-琥珀酰亚胺基-4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯(SMCC),一种异双功能交联剂,在PEI和含硫醇基团的肽之间形成稳定的键。