Sequential Adeno-Associated Viral Vector Serotype 9–Green Fluorescent Protein Gene Transfer Causes Massive Inflammation and Intense Immune Response in Rat Striatum

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

Download Full-text

Stress-survival responses of a carbon-starvedp-nitrophenol-mineralizingMoraxellastrain in river water

Canadian Journal of Microbiology ◽

10.1139/w04-131 ◽

2005 ◽

Vol 51 (3) ◽

pp. 223-229 ◽

Cited By ~ 2

Author(s):

Michael Moore ◽

Jack Trevors ◽

Hung Lee ◽

Kam Tin Leung

Keyword(s):

Green Fluorescent Protein ◽

River Water ◽

Water Samples ◽

Protein Gene ◽

Fluorescent Protein ◽

Carbon Starvation ◽

Green Fluorescent Protein Gene ◽

River Water Samples ◽

Stress Survival ◽

Green Fluorescent

The effect of carbon starvation on the stress-resistant responses of a p-nitrophenol-mineralizing Moraxella strain was examined in both buffer and river water samples. The Moraxella strain showed optimal stress-resistant responses in a minimal salt buffer when carbon-starved for 1–2 d. In the buffer system, the 1- and 2-day carbon-starved Moraxella cultures survived about 150-, 200-, and 100-fold better than the non-starved cultures when exposed to 43.5 °C, 2.7 mol/L NaCl, and 500 µmol/L H2O2for 4 h, respectively. A green fluorescent protein gene- (gfp) labelled derivative of the Moraxella strain was used to examine the stress-resistant responses of the bacterium in natural river water microcosms. The carbon-starved gfp-labelled Moraxella strain also showed stress-resistant responses against heat, osmotic, and oxidative stresses in the river water samples. Despite the stress-tolerant capability of the carbon-starved gfp-labelled Moraxella cells, they did not exhibit any survival advantage over their non-starved counterparts when inoculated into river water microcosms and incubated at 10 and 22 °C for 14 d.Key words: carbon starvation, stress-survival responses, Moraxella, p-nitrophenol, green fluorescent protein gene.

Download Full-text