title: Small Interfering RNA-Mediated Connexin Gene Knockdown in Vascular Endothelial and Smooth Muscle Cells.
abstract: Global knockout of vascular connexins can result in premature/neonatal death, severe developmental complications, or compensatory up-regulation of different connexin isoforms. Thus, specific connexin gene knockdown using RNAi-mediated technologies is a technique that allows investigators to efficiently monitor silencing effects of single or multiple connexin gene products. The present chapter describes the transient knockdown of connexins in vitro and ex vivo for cells of the blood vessel wall. In detail, different transfection methods for primary endothelial cells and ex vivo thoracodorsal arteries are described. Essential controls for validating transfection efficiency as well as targeted gene knockdown are explained. These protocols provide researchers with the ability to modify connexin gene expression levels in a multitude of experimental setups.
authors.affiliation: Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
authors.forename: Miranda E
authors.initials: ME
authors.lastname: Good
authors.affiliation: Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Begandt
authors.affiliation: Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.|Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
authors.forename: Leon J
authors.initials: LJ
authors.lastname: DeLalio
authors.affiliation: Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
authors.forename: Scott R
authors.initials: SR
authors.lastname: Johnstone
authors.affiliation: Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. brant@virginia.edu.|Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. brant@virginia.edu.
authors.forename: Brant E
authors.initials: BE
authors.lastname: Isakson
mesh_terms: D000818:Animals; D001158:Arteries / Q000378:metabolism*; D002478:Cells, Cultured; D017630:Connexins / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D061251:Primary Cell Culture / Q000379:methods; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D046509:Tissue Culture Techniques / Q000379:methods; D014162:Transfection / Q000379:methods; D015854:Up-Regulation
chemical_list: D017630:Connexins; D034741:RNA, Small Interfering
keywords: Electroporation; Ex vivo transfection; Knockdown; Lipofection; Off-target; Transfection; siRNA