Efficient protein transfection of cultured coronary venular endothelial cells
- PMID: 9815096
- DOI: 10.1152/ajpheart.1998.275.5.H1873
Efficient protein transfection of cultured coronary venular endothelial cells
Abstract
Although it is well recognized that microvascular endothelial cells play an important role in the local regulation of tissue perfusion and exchange processes, the precise effect of specific endothelial proteins on microvascular function remains to be elucidated. The lack of information is partially due to methodological limitations, because pharmacological approaches that are routinely used in conventional microcirculatory studies produce nonspecific information. The purpose of this study was to develop an efficient method of transfecting endothelial cells with proteins for functional analysis. TransIT, a polyamine reagent, proved very successful for beta-galactosidase (beta-Gal) protein transfection of bovine coronary venular endothelial cells, because time-course and dose-dependent experiments showed that a transfection efficiency of 88 +/- 7% was possible. In control studies, beta-Gal was detected in transfected cells that were trypsinized and washed, indicating that the protein was not merely adhering to the cell surface. Furthermore, transfection of a cell-impermeable peptide inhibitor of protein kinase C (PKC) resulted in a decrease in PKC activity in comparison with control cells. This approach provides a technical basis for further transfection of endothelial cell monolayers with antibodies and constitutively active or dominant-negative proteins to study the molecular control of microvascular function.
Similar articles
-
Cell-selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting.FASEB J. 2001 Feb;15(2):416-26. doi: 10.1096/fj.00-0022com. FASEB J. 2001. PMID: 11156957
-
Focal adhesion kinase in neutrophil-induced microvascular hyperpermeability.Microcirculation. 2005 Mar;12(2):223-32. doi: 10.1080/10739680590905251. Microcirculation. 2005. PMID: 15824042
-
High efficiency gene transfer by multiple transfection protocol.Histochem J. 1999 Apr;31(4):241-3. doi: 10.1023/a:1003598614323. Histochem J. 1999. PMID: 10447066
-
Protein transfection of intact microvessels specifically modulates vasoreactivity and permeability.J Vasc Res. 2001 Sep-Oct;38(5):444-52. doi: 10.1159/000051077. J Vasc Res. 2001. PMID: 11561146
-
How to study dendriplexes II: Transfection and cytotoxicity.J Control Release. 2010 Jan 25;141(2):110-27. doi: 10.1016/j.jconrel.2009.09.030. Epub 2009 Oct 6. J Control Release. 2010. PMID: 19815039 Review.
Cited by
-
Delivery of antibodies to the cytosol: debunking the myths.MAbs. 2014 Jul-Aug;6(4):943-56. doi: 10.4161/mabs.29268. Epub 2014 May 21. MAbs. 2014. PMID: 24848507 Free PMC article.
-
A practical approach for intracellular protein delivery.Cytotechnology. 2008 Jan;56(1):41-8. doi: 10.1007/s10616-007-9102-3. Epub 2007 Oct 16. Cytotechnology. 2008. PMID: 19002840 Free PMC article.
-
Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor.J Physiol. 2003 Nov 1;552(Pt 3):691-9. doi: 10.1113/jphysiol.2003.048405. Epub 2003 Aug 29. J Physiol. 2003. PMID: 12949227 Free PMC article.
-
Transference of recombinant VE-cadherin cytoplasmic domain alters endothelial junctional integrity and porcine microvascular permeability.J Physiol. 2004 Jan 1;554(Pt 1):78-88. doi: 10.1113/jphysiol.2003.051086. J Physiol. 2004. PMID: 14678493 Free PMC article.
-
Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells.Mol Ther Methods Clin Dev. 2018 Dec 20;13:99-111. doi: 10.1016/j.omtm.2018.12.005. eCollection 2019 Jun 14. Mol Ther Methods Clin Dev. 2018. PMID: 30740472 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
