Applications of green fluorescent protein as a marker of retroviral vectors

Abstract

The Green Fluorescent Protein (GFP) of Aequorea victoria is used as a vital fluorescent tag for the detection and isolation of genetically modified cells. Several modified variants of GFP were tested as marker genes in retroviral vectors containing different backbones and promoter combinations. Constructs allowing for reliable detection of GFP fluorescence and the expression of a cotransduced gene from a strong promoter were identified. Cells harboring such constructs are detectable by flow cytometry, fluorescence microscopy and multi-well fluorescence reading. GFP expression in transduced cells is stable both in vitro and in vivo, and long-term dynamics of GFP-positive fractions in a mixed population can be used to monitor the biological effects of a cotransduced gene. Selection of cells with the highest GFP fluorescence enriches for multiply infected cells. The use of different GFP variants allows one to monitor simultaneously two cell populations transduced with vectors carrying GFPs that differ in their fluorescence intensity or spectral properties and to identify doubly transduced cells. In addition, transcription of an inducible promoter positioned in the opposite orientation to GFP can be monitored by the inhibition of GFP fluorescence. Thus, GFP provides a useful marker for gene transfer by retroviral vectors and extends the range of applications for retroviral transduction.

Apoptosis Inhibitor as a Suppressor of Tumor Progression: Expression of Bcl-2 Eliminates Selective Advantages for p53-Deficient Cells in the Tumor

“…Hamster and mouse cells were transduced with retroviral vector pLNCgE, carrying cDNA of green fluorescent protein (GFP) under the control of CMV promoter 18 with subsequent sorting of highly fluorescent cells using FACSorter (Beckton Dickinson).…”

p21Waf1/Cip1/Sdi1 mediates retinoblastoma protein degradation

“…To verify that this effect of GSE56 was directly attributable to p21, we developed a derivative of HT1080 cells with (Chang et al, 1999) (left lanes) or HT1080 p21-9 transduced with p53 inhibiting peptide GSE56 (right lanes) after the addition of 50 mM IPTG (Sigma, St Louis, MO, USA). GSE56 (Ossovskaya et al, 1996) was introduced into HT1080 p21-9 cells via a retroviral vector LXSE (Kandel et al, 1997); the transduced cells were selected by flow sorting for green fluorescent protein fluorescence. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FC2 serum.…”

Transposon-based mutagenesis identifies short RIP1 as an activator of NFκB

“…Flow cytometry was done as described. 46 NFκB dependence test. C6TA4 and 3B4.1 cells were infected with pBabePuro 47 or pBabePuro-srIκB (a gift of Dr. S. Makarov, UNCCH) as described.…”

“…C6TA4 and 3B4.1 cells were infected with pBabePuro 47 or pBabePuro-srIκB (a gift of Dr. S. Makarov, UNCCH) as described. 46 Each infected culture was split into two (with or without TNFα treatment) and selected for puromycin resistance.…”