Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo.
Expression of a fluorescent reporter gene has been studied using two alternate promoters to transcribe the green fluorescent protein (gfp) from Aequorea victoria. The human cytomegalovirus (CMV) enhancer/ promoter or the human muscle-specific creatine kinase promoter (CKM) were inserted along with the gfp cDNA into a plasmid expression vector based on a modified adeno-associated virus genome. Naked plasmid DNA was injected into the hamstring muscle of mdx mice and gfp gene expression determined from frozen muscle sections taken at 4, 14, and 42 days postinjection. Fluorescence patterns obtained by photomicroscopy and quantitative fluorescence measurements indicated a near-linear increase in the accumulation of the gfp in skeletal muscle during the length of the study, with gfp expression at 42 days being roughly four times the values obtained at 4 days. The levels of expression of gfp from the CKM construct were consistantly higher than for the CMV construct. The CKM promoter/expression vector combination demonstrates significant potential for simple, direct delivery and long-term, high-level expression of genes in skeletal muscle.
Bartlett
R J
RJ
Department of Neurology, University of Miami School of Medicine, FL 33136, USA. rbartlet@mednet.med.miami.edu
Secore
S L
SL
Singer
J T
JT
Bodo
M
M
Sharma
K
K
Ricordi
C
C
D000818:Animals; D001483:Base Sequence; D017690:Cell Transplantation; D003402:Creatine Kinase / Q000096:biosynthesis* / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics*; D017931:DNA Primers; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000637:transplantation*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D003402:Creatine Kinase