Wickersham Ian R, Sullivan Heather A, Pao Gerald M, Hamanaka Hiroki, Goosens Ki A, Verma Inder M, Seung H Sebastian
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;
Cold Spring Harb Protoc. 2015 Apr 1;2015(4):368-74. doi: 10.1101/pdb.prot075879.
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.
用狂犬病病毒（RV）包膜糖蛋白假型化的慢病毒载体可通过轴突终末有效感染，从而将转基因稳定递送至投射到注射部位的远处神经元，但对于大多数神经科学应用而言，其产生的表达水平过低且变化不定。然而，如果用于递送重组酶或反式激活因子，慢病毒载体在报告基因小鼠中使用或与第二种病毒联合使用以高水平表达“有效载荷”转基因时，是靶向投射神经元的极佳手段。对于大量神经元的逆行感染，高病毒滴度至关重要。在此，我们展示了用于生成高滴度上清液的试剂和方案，这些上清液可浓缩1000倍，最终滴度超过每毫升10(10)个感染单位。我们通过在敲入报告基因小鼠中选择性靶向皮质丘脑和皮质顶盖神经元以实现荧光团的高水平表达，证明了这些载体的实用性。
