title: A vector-host system to fingerprint virus tropism.
abstract: Reporter genes are important tools for assessing vector pharmacology in vivo. Although useful, current systems are limited by (1) the need to generate a new vector for each different reporter, (2) the inability to package reporter genes in small vectors, and (3) variations in reporter gene feedback due to variations in cell-to-cell vector copy number. To circumvent these problems, we have used Cre recombinase as a "cat's paw" to activate reporter genes embedded in transgenic mice. The small Cre gene was introduced into self-complementary adeno-associated viral (scAAV) vectors with limited packaging capacity. Injection of scAAV-Cre vectors into mice with loxP-inactivated luciferase enabled in vivo imaging distributions comparable to the signal observed after AAV-luciferase injection. When injected into mT/mG mice, AAV-Cre converted ubiquitous expression of red fluorescent protein (RFP) to green fluorescent protein (GFP) expression only where the vectors transduced cells. Injection into F(1) hybrid luciferase and mT/mG mice enabled simultaneous three-reporter tracking. This system was able to discriminate cell-specific transduction in all organs tested, with particular usefulness for detecting AAV serotype-specific transduction in the liver, kidney, and muscle. Given that F(1) mice bear exactly one copy of luciferase and one copy of RFP-GFP, each reporter gene is either "on" or "off" in a cell. The Cre system therefore provides a unique quantum method to quantify vector delivery that can be applied when vector capacity is limited.
authors: Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
authors: Matthew L
authors: ML
authors: Hillestad
authors: Adam J
authors: AJ
authors: Guenzel
authors: Karl A
authors: KA
authors: Nath
authors: Michael A
authors: MA
authors: Barry
mesh_terms: D000818:Animals; D003062:Codon / Q000235:genetics; D003433:Crosses, Genetic; D000229:Dependovirus / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D012703:Serotyping; D014161:Transduction, Genetic; D056189:Viral Tropism / Q000235:genetics*
chemical_list: D003062:Codon; D008156:Luciferases; C045073:Cre recombinase; D019426:Integrases