title: High efficiency transfection of embryonic limb mesenchyme with plasmid DNA using square wave pulse electroporation and sucrose buffer.
abstract: 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.
authors: Department of Health and Human Services, Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD.
authors: Brent E
authors: BE
authors: Bobick
authors: Department of Health and Human Services, Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD; Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, PA.
authors: Peter G
authors: PG
authors: Alexander
authors: Department of Health and Human Services, Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD; Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, PA.
authors: Rocky S
authors: RS
authors: Tuan
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000378:metabolism*; D018274:Electroporation / Q000379:methods*; D008648:Mesoderm / Q000166:cytology*; D010957:Plasmids / Q000235:genetics; D013395:Sucrose / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
chemical_list: D013395:Sucrose; D004247:DNA
keywords: cell suspension; chondrogenesis; embryonic limb mesenchyme; micromass culture; square wave pulse electroporation; sucrose buffer; transfection protocol