For example, DNA viruses, such as PRV, can be readily recovered following manipulation of the viral genome and expression of encoded genes can be made conditional upon interaction with recombinase Lumacaftor expressed in transgenic mouse lines (DeFalco et al., 2001). In contrast, recovery of new rabies virus variants requires a more complex process (Inoue et al., 2003, Ito et al., 2003, Schnell et al., 1994 and Wu and Rupprecht, 2008), and the ability to interface with transgenic mice requires development of novel strategies, as described below. Nevertheless, once a new ΔG rabies virus variant is successfully recovered, it can easily be propagated and amplified in a rabies glycoprotein-expressing cell line (Etessami et al., 2000,

Mebatsion et al., 1996, Wickersham et al.,

2007a and Wickersham et al., 2007b). Here we establish reliable and efficient methods and reagents for recovery and amplification of rabies virus and describe the development and validation of the new SADΔG variants that we have produced. These variants include SADΔG rabies viruses encoding red fluorescent proteins; blue fluorescent proteins; both red and green fluorescent proteins from the same genome; the calcium sensor GCaMP3 for monitoring neuronal activity (Tian et al., 2009); the light-gated cation channel channelrhodposin-2 (ChR2) for the activation of neural activity (Boyden et al., selleck products 2005); the Drosophila allatostatin receptor (AlstR) for silencing of neural activity ( Lechner et al., 2002 and Tan et al., 2006); and the reverse tetracycline transactivator (rtTA), tamoxifen-inducible Cre-recombinase, and flippase (FLP)-recombinase to allow control Cell press of gene expression in available transgenic mouse lines and viral vectors ( Branda and Dymecki, 2004). We illustrate the utility of these variants and further discuss an even wider potential range of powerful applications. Although SADΔG-GFP can be recovered from DNA plasmids and amplified

using previously established procedures (Buchholz et al., 1999), we aimed to improve the efficiency of ΔG rabies virus recovery from DNA. Here, we generated new plasmids and cell lines and tested various culture conditions to optimize recovery systems. Because rabies is a negative strand RNA virus but the tools that are available to manipulate genetic material work with DNA, it is necessary to generate and use several specialized reagents in order to recover new genetically-modified rabies variants from a set of DNA plasmids. At least four different groups have developed and used such systems to recover various rabies strains (Inoue et al., 2003, Ito et al., 2003, Schnell et al., 1994 and Wu and Rupprecht, 2008). The first published system recovered the SAD-B19 strain of rabies virus, utilized transcription from the T7 promoter, required T7 RNA polymerase provided by a vaccinia helper virus (Schnell et al., 1994), and in later developments was supported by a cell line expressing T7 polymerase (BSR T7/5) (Buchholz et al., 1999 and Wickersham et al.