Zolotukhin S, Potter M, Hauswirth W W, Guy J, Muzyczka N
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610, USA.
J Virol. 1996 Jul;70(7):4646-54. doi: 10.1128/JVI.70.7.4646-4654.1996.
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.
我们构建了gfph，它是水母维多利亚多管发光水母绿色荧光蛋白（gfp）cDNA的合成版本，适用于在哺乳动物细胞，尤其是人类来源的细胞中高水平表达。为了改变gfp10编码序列内的密码子使用情况，使其更适合在哺乳动物细胞中表达，我们在88个密码子中总共进行了92个碱基替换。我们还描述了一系列通用的重组腺相关病毒和腺病毒载体，用于将基因传递并表达于哺乳动物细胞中，并利用这些载体证明了gfph基因在人细胞系293中以及在豚鼠眼的神经感觉细胞内的高效转导和表达。感染重组腺相关病毒-GFPH的细胞可以通过荧光激活细胞分选轻松分选，这表明新设计的gfph基因可广泛用作许多基因传递技术（包括人类基因治疗）中的报告基因。
