Liposome-mediated transfection of mature taste cells.
The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used alpha-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common "promiscuous" promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells.
Landin
Ana Marie
AM
Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136, USA.
Kim
Joung Woul
JW
Chaudhari
Nirupa
N
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000378:metabolism; D002478:Cells, Cultured; D005047:Etoposide / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D007150:Immunohistochemistry / Q000379:methods; D007527:Isoenzymes / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008130:Lomustine / Q000378:metabolism; D008164:Luminescent Proteins / Q000096:biosynthesis; D008297:Male; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D009474:Neurons / Q000145:classification / Q000502:physiology*; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D054799:Phospholipase C beta; D051381:Rats; D017207:Rats, Sprague-Dawley; D013650:Taste Buds / Q000166:cytology* / Q000187:drug effects; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000378:metabolism
D007527:Isoenzymes; D008081:Liposomes; D008164:Luminescent Proteins; D019006:Neural Cell Adhesion Molecules; D020648:Peptide Elongation Factor 1; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D005047:Etoposide; D008130:Lomustine; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C489034:UCHL1 protein, rat; D043222:Ubiquitin Thiolesterase; D008727:Methotrexate