CMV-GFP lentiviral particles

Viral vectors are frequently used to deliver and direct the expression of transgenes in a spatially and temporally restricted manner within the nervous system of a variety of model organisms. Although viral vectors are commonly used to direct ectopic expression of transgenes within the nervous system, creating high-titer viral vectors capable of expressing very large transgenes or difficult-to-express transgenes presents unique challenges. Here, researchers created a number of custom-designed AAV and lentiviral vectors optimized for large transgenes by minimizing unnecessary DNA sequences, utilizing short promoter sequences from eight widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin, and CMV), and utilizing very short (~75 bps) 3′ untranslated sequences. Not surprisingly, these promoters differed in their ability to express GluN2 subunits. Surprisingly, however, the neuron-specific synapsin and αCaMKII promoters failed to confer detectable expression of the full-length GluN2 subunit, and detectable expression was achieved from these promoters only when the transgene contained an intron or the GluN2 subunit transgene was truncated to contain only the coding region of the GluN2 transmembrane domain.

Here, it was shown that the TRE3G-GluN2A, TRE3G-GluN2B, CMV-GluN2A, CMV-GluN2B, TRE3G-GFP, and CMV-GFP lentiviruses exhibited convincing robust transgene expression (Figure 10). Since lentiviruses are very efficient at transducing non-neuronal cells in vivo, the CMV-GluN2A, CMV-GluN2B lentiviruses are not suitable for in vivo use, as the transgene will be robustly expressed in non-neuronal cells. This is unfortunate, considering that they do function as intended. However, the TRE3G-GluN2A and TRE3G-GluN2B lentiviruses also function as intended, and if these viruses are used in αCaMKII-tTA transgenic mice, the GluN2 transgene will be specifically expressed in αCaMKII-positive neurons. The researchers found that the lentiviruses could transduce 293FT cells very efficiently, but the lentiviruses did not transduce N2A cells well, so lentiviruses containing transgenes controlled by the synapsin or αCaMKII promoters could not be tested in cell culture—these were tested in vivo.

Figure 1. GluN2 and GFP transgene expression in vitro mediated by lentivirus. (Holehonnur R, et al., 2015)