A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells.
We constructed gfph, a synthetic version of the jellyfish Aequorea victoria green fluorescent protein (gfp) cDNA that is adapted for high-level expression in mammalian cells, especially those of human origin. A total of 92 base substitutions were made in 88 codons in order to change the codon usage within the gfp10 coding sequence so that it was more appropriate for expression in mammalian cells. We also describe a series of versatile recombinant adeno-associated virus and adenovirus vectors for delivery and expression of genes into mammalian cells and, using these vectors, demonstrate the efficient transduction and expression of the gfph gene in the human cell line 293 and also in vivo, within neurosensory cells of guinea pig eye. Cells infected with recombinant adeno-associated virus-GFPH can be readily sorted by fluorescence-activated cell sorting, suggesting that the newly designed gfph gene could be widely used as a reporter in many gene delivery technologies, including human gene therapy.
Zolotukhin
S
S
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610, USA.
Potter
M
M
Hauswirth
W W
WW
Guy
J
J
Muzyczka
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006168:Guinea Pigs; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D012160:Retina / Q000378:metabolism; D037401:Scyphozoa
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins