In vitro expression of calcitonin gene-related peptide in human endothelial cells transfected with plasmid and retroviral vectors.
Introduction of cDNA encoding human alpha-calcitonin gene-related peptide (CGRP), a potent vasodilator, into cultured human umbilical vein endothelial cells (HUVEC) was undertaken by using plasmids and retroviral vectors. In order to improve expression, modification of context coding sequence for the initiation of CGRP translation and deletion of nontranslated regions of CGRP cDNA in the transfection vectors were tested. Stable transfer of neo in the HUVEC was achieved with both plasmid and retroviral vectors. Integration rates obtained by using retrovirus (approximately 1%), where higher than those achieved with plasmid-mediated transfection (< 1/1000). CGRP expressed in the transfected HUVEC was secreted into culture medium when a leading sequence was included in the expression vectors. CGRP was detected by enzyme-linked immunosorbent assay (ELISA) in the supernatants of both transiently transfected and stably transfected/infected HUVEC. Higher levels of expression were achieved by using plasmid (giving a maximum CGRP concentration of 6.5 +/- 0.5 pM in the supernatant) than retroviruses. Lipofectin-mediated transfer of CGRP cDNA also resulted in transient expression of CGRP in the HUVEC.
Sun
B
B
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, UK.
Plumpton
C
C
Sinclair
J H
JH
Brown
M J
MJ
D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D018076:DNA, Complementary; D015740:Calcitonin Gene-Related Peptide