A Aints1, M S Dilber, C I Smith. 1. Department of Medicine, Clinical Research Centre, Huddinge Hospital, Karolinska Institutet, Sweden.
Abstract
BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 polypeptide has been reported to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus, a feature making it a useful tool in bystander cell-targeted gene therapy. METHODS: Here we show, by detection of Green Fluorescent Protein (GFP) fused to VP22, its subcellular distribution in living producer (transfected) and recipient (non-transfected) cells as well as in cells after fixation. Four cell lines from different species were used. RESULTS: Different fractions of translocated GFP-VP22 fusion protein could be detected in fixed recipient cells by two different methods of fixation. Functional GFP in live recipient cells could not be detected. CONCLUSIONS: Our study indicates that the VP22-chimeric protein transfer in its present form is suboptimal in terms of protein function. However, after fixation, the GFP signal in 100% of cells in a monolayer can be detected even at moderate transfection efficiency.
BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 polypeptide has been reported to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus, a feature making it a useful tool in bystander cell-targeted gene therapy. METHODS: Here we show, by detection of Green Fluorescent Protein (GFP) fused to VP22, its subcellular distribution in living producer (transfected) and recipient (non-transfected) cells as well as in cells after fixation. Four cell lines from different species were used. RESULTS: Different fractions of translocated GFP-VP22 fusion protein could be detected in fixed recipient cells by two different methods of fixation. Functional GFP in live recipient cells could not be detected. CONCLUSIONS: Our study indicates that the VP22-chimeric protein transfer in its present form is suboptimal in terms of protein function. However, after fixation, the GFP signal in 100% of cells in a monolayer can be detected even at moderate transfection efficiency.
Authors: Maria Teresa Sciortino; Brunella Taddeo; Maria Giuffrè-Cuculletto; Maria Antonietta Medici; Antonio Mastino; Bernard Roizman Journal: J Virol Date: 2007-08-01 Impact factor: 5.103
Authors: Brunella Taddeo; Maria Teresa Sciortino; Weiran Zhang; Bernard Roizman Journal: Proc Natl Acad Sci U S A Date: 2007-07-09 Impact factor: 11.205