Retroviral transfer and expression of a humanized, red-shifted green fluorescent protein gene into human tumor cells

Abstract

Over two-thirds of the current gene therapy protocols use retroviral gene transfer systems. We have developed an efficient retroviral-based method that allows rapid identification of gene transfer in living mammalian cells. Cells were generated containing a gene for an improved (humanized, red-shifted) version of the Aequorea victoria green fluorescent protein (hRGFP) from a retroviral vector. The hRGFP gene was used to produce an amphotropic vector producer cell line that demonstrated vibrant green fluorescence after excitation with blue light. A375 melanoma cells transduced with the retroviral vector demonstrated stable green fluorescence. Both PA317 murine fibroblasts and A375 human cell lines containing the vector were easily detected by FACS analysis. These vectors represent a substantial improvement over currently available gene transfer marking systems. Bright, long-term expression of the hRGFP gene in living eukaryotic cells will advance the study of gene transfer, gene expression, and gene product function in vitro and in vivo particularly for human gene therapy applications.

Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters

“…22 These genes are flanked by the 145 bp terminal repeats ticle per cell, we used the SCFA to compare its efficiency to the ICA. 22,39 293 Cells in a 96-well dish, plated at a 50% of the adeno-associated virus. This vector was digested with KpnI, blunt-ended with T 4 polymerase, and partially confluency, were infected with serial dilutions of rAAV-UF5 and co-infected with Ad (MOI of 10) to increase the digested with XhoI to remove the CMV promoter segment.…”

Pure populations of transduced primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry

“…However, much interest has recently been shown in green fluorescent protein (GFP) as a marker as it allows expression monitoring in live cells, 7 and GFP expressing retro-and adenovirus vectors have been reported. 8,9 The availability of newer mutants of GFP exhibiting enhanced fluorescence characteristics has considerably improved b a…”

Green Fluorescent Protein as a Selectable Marker of Retrovirally Transduced Hematopoietic Progenitors

“…Murine stem/progenitor cells were transducible with a GFP-gene-containing retrovirus, and the successfully transduced cells were readily separated. Others have recently shown transduction of GFP genes into a variety of cells, including the hematopoietic system [17][18][19][20][21]. Compared to a study by Persons et al [19], in which murine BM cells were transduced more efficiently (about 40%), our transduction frequency was low (up to 5%).…”

“…As has been documented, strong internal promoters may interfere with transcription from the proviral LTR and compromise viral titers, and incorporation of introns into retroviral constructs may further complicate the titer problem. Recent advances in engineering GFP genes allowed brighter fluorescence and more efficient translation in mammalian cells [18,22]. We are currently evaluating simpler retrovirus constructs containing humanized GFP genes, and some of these vectors actually achieved higher titers (unpublished).…”