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. Author manuscript; available in PMC: 2008 Dec 15.
Published in final edited form as: Gene. 2007 Sep 19;405(1-2):65–78. doi: 10.1016/j.gene.2007.09.010

Fig. 1. Generation of recombinant lentiviruses and HIV-1 reporter viruses.

Fig. 1

(A) For lentiviral production, 293T cells are transfected via the calcium phosphate method, with a packaging construct that directs the expression of viral proteins minus the Env and Vpu, a plasmid encoding the VSV-G envelope and a transfer plasmid that contains the genes of interest under the control of CMV promoters (expression cassettes) similarly as described previously (Naldini et al., 1996; Hasham and Tsygankov, 2004). In the transfer constructs (pCPP or pCEIII), the indicated expression cassettes are flanked by HIV-1 LTRs and contain the packaging signal, as well as other indicated elements required for efficient lentiviral packaging. Supernatants are collected 48 and 60 hours later and concentrated by ultracentrifugation. cDNAs encoding HA-dnCDK9, HA-CDK9, and HA-cyclin T1 were subcloned in pCPP. Additionally, HA-dnCDK9 and Flag-Hexim1 were subcloned in pCEIII transfer vectors as described in the Methods section. Gene X represents HA-dnCDK9, HA-CDK9, HA-cyclin T1 or Flag-Hexim1. (B) HIV-1-luc viruses pseudotyped with either HXB2 or VSV-G envelopes are generated by cotransfection of the HIV-1 molecular clone mutant NL4-3.Luc.R-E- and the indicated envelope plasmids in 293T cells. These viruses are fully infectious and are replication defective as they exhibit a mutation in the Env and Vpr genes. Luciferase expression is directly proportional to transcription of full-length HIV-1 genome and, thus, reflects replication (Connor et al., 1995; He et al., 1995).