Rapid in vivo isolation of gene expression elements using an HSV amplicon system.
Short-lived gene expression elements (GEE) represent currently a significant obstacle for gene therapy. To identify GEE such as promoters, enhancers, locus control regions, or insulators, useful for long-term or tissue-specific gene therapy, we developed a GEE trapping strategy in which any sequence can be screened for activity in vivo and the expressing clones can be rapidly isolated. Test sequences are introduced into a herpesvirus (HSV) amplicon vector that expresses green fluorescent protein (GFP) only if the insert has GEE function. The plasmid amplicons can be packaged and used to transduce either cultured cells or any tissue or organ in vivo. Single cell suspensions can then be prepared and GFP positive cells isolated by FACS. After sorting, the plasmid amplicons can be isolated and reintroduced into bacteria, cloning the GEE for further characterization. The CMV promoter was used to demonstrate the utility of the system. The amplicon vector was packaged into herpesvirus virions and transduced into Vero cells, confirming the vector can be packaged. After injection into rat eyes, the packaged amplicon virions were capable of transducing cells and the GFP expressing plasmid amplicons were recovered from rat eye tissues by single cell isolation followed by FACS. This novel amplicon system should prove valuable in identifying and characterizing GEE for use in gene therapy.
D000818:Animals; D019677:Artificial Gene Fusion; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D018139:Simplexvirus / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection; D019065:Virus Assembly
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins