Biología molecular: acceso abierto

An Alternate Method for Efficient Delivery of Catalyzing Enzymes

Abstract

www.omicsonline.org/clinical-experimental-ophthalmology.phpUma Lakshmipathy, Andrew Fontes, Michael Poderycki and Jonathan D. Chesnut

Improved cellular engineering tools have enabled scientists to modify genomic sequences at a new pace for a variety of applications. However, in order to create more accurate tools for site specific targeting and editing, improved delivery systems for modifying enzymes are required. These enzymes allow end users to disrupt, insert or edit genes utilizing sequence specific homology. Current applications require co-transfection of an engineering plasmid in tandem with the vector carrying the modifying enzyme. However, co-transfection can negatively affect transfection efficiencies as well as increase probability of randomly integrating DNA. Here we report the transient delivery of catalyzing enzymes via BacMam non-integrating virus at high efficiency. Baculovirus is a non-infectious delivery system that can transduce a variety of mammalian cells. We have shown improved delivery of NLS-Cre recombinase for the excision of regions in disrupted genes to enable expression. In addition, we have tested PhiC31 integrase delivery for the guided insertion of plasmid DNA to a specific locus. The transient expression provided by BacMam, coupled with ease of transfection, creates an ideal system for delivery of catalyzing enzymes in mammalian cells.