Open AccessHigh efficiency transfection of embryonic limb mesenchyme with plasmid DNA using square wave pulse electroporation and sucrose buffer
Author(s) -
Brent E. Bobick,
Peter G. Alexander,
Rocky S. Tuan
Publication year - 2014
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000114136
Subject(s) - chondrogenesis , mesenchyme , transfection , electroporation , microbiology and biotechnology , biology , embryonic stem cell , limb bud , reporter gene , cartilage , cell culture , embryo , anatomy , genetics , stem cell , gene expression , gene
Micromass cultures of primary embryonic limb mesenchyme are a valuable model system for studying cartilage formation in vitro. However, high efficiency introduction of plasmid DNA into this hard-to-transfect cell type typically results in considerable cell death and significantly impeded chondrogenesis when the cells are subsequently plated in high density micromass. Here, we describe a novel method in which square wave pulse electroporation of chick embryo wing bud mesenchyme suspended in protective sucrose buffer results in high efficiency transfection without substantially affecting micromass culture cell viability or chondrogenic differentiation potential. Furthermore, we show that this protocol can be employed, along with effector gene expression vectors, to generate observable changes in the amount of cartilage tissue formed in micromass, unlike lower efficiency, higher cytotoxicity techniques that require co-transfection of reporter plasmids to monitor changes in the extent of chondrogenesis and correct for differences in cell viability.