title: Lentiviral vectors for retrograde delivery of recombinases and transactivators.
abstract: Lentiviral vectors pseudotyped with the rabies virus (RV) envelope glycoprotein efficiently infect via axon terminals to stably deliver transgenes to distant neurons projecting to an injection site, but the resulting expression levels are too low and variable for most neuroscientific applications. If used to deliver recombinases or transactivators, however, lentiviral vectors are excellent means of targeting projection neurons when used in reporter mice or in combination with a second virus to express "payload" transgenes at high levels. For retrograde infection of significant numbers of neurons, high virus titers are critical. Here we present reagents and a protocol for generating high-titer supernatants that can be concentrated 1000-fold for final titers in excess of 10(10) infectious units per milliliter. We demonstrate the usefulness of these vectors by selectively targeting corticothalamic and corticotectal neurons for high-level expression of a fluorophore in knock-in reporter mice.
authors.affiliation: McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: Ian R
authors.initials: IR
authors.lastname: Wickersham
authors.affiliation: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: Heather A
authors.initials: HA
authors.lastname: Sullivan
authors.affiliation: Laboratory of Genetics-V, Salk Institute for Biological Studies, La Jolla, California 92037.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Pao
authors.affiliation: McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hamanaka
authors.affiliation: McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: Ki A
authors.initials: KA
authors.lastname: Goosens
authors.affiliation: Laboratory of Genetics-V, Salk Institute for Biological Studies, La Jolla, California 92037.
authors.forename: Inder M
authors.initials: IM
authors.lastname: Verma
authors.affiliation: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: H Sebastian
authors.initials: HS
authors.lastname: Seung
mesh_terms: D005822:Genetic Vectors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000378:metabolism*; D045522:Recombinases / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014461:Ultracentrifugation
chemical_list: D045522:Recombinases; D015534:Trans-Activators