Intra-Molecular Homologous Recombination of Scarless Plasmid

Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.

A gene-activating skin substitute comprising PLLA/POSS nanofibers and plasmid DNA encoding ANG and bFGF promotes in vivo revascularization and epidermalization

A gene-activated porous nanofibrous scaffold for effectively promoting vascularization, epidermalization and dermal wound healing by sustained release of dual plasmid DNAs.

A biophysical study of gene nanocarriers formed by anionic/zwitterionic mixed lipids and pillar[5]arene polycationic macrocycles

A multivalent cationic macrocycle is used as a mediator between plasmid DNAs and anionic lipids (ALs) to build an efficient and safe gene nanocarrier.