( e as plasmid DNA and as a tr...)New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence ,,, Humanized Renilla GFP Reporter

e as plasmid DNA and as a transduction-ready, replication-incompetent, vesicular stomatitis virus (VSV)-G pseudotyped high-titer retroviral supernatant. In plasmid form, the pFB-hrGFP retroviral vector must be packaged into infectious virion particles.³ We recommend using Stratagenes Vpack vectors which consist of a set of 5 vectors that can be used with any MMLV-based retroviral vector to produce viral supernatants in transient triple-transfection experiments. The vectors include a gag-pol-expressing vector that is cotransfected with the retroviral expression vector together with one of a choice of 4 envelope (env)-expressing vectors. The choice of the env-expressing vector is based on the range of cell types the user wishes to transduce. With the pVPack vector system, all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, with minimal or no sequence overlap between the plasmids, resulting in the generation of replication-incompetent virions. As an alternative strategy, Stratagene also offers a pFB-hrGFP transduction-ready supernatant that may be used to immediately deliver the hrGFP gene to cells, thereby eliminating the process of generating infectious virus particles. The supernatants are titered for ease in controlling the desired multiplicity of infection.

Fig.5

To test the performance of our pFB-hrGFP retroviral vector, we used this vector to transduce several widely used mammalian cell types. Figure 5 illustrates the high-intensity fluorescence that resulted upon delivery of hrGFP to human, rodent, and simian cells. To further demonstrate the high transduction efficiency of this retroviral vec
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