Generation of a Stable Cell Line Producing High-Titer Self-Inactivating Lentiviral Vectors  Kailin Xu, Hong Ma, Thomas J. McCown, Inder M. Verma, Tal Kafri  Molecular Therapy  Volume 3, Issue 1, Pages 97-104 (January 2001) DOI: 10.1006/mthe.2000.0238 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Features of the conditional SIN lentivirus vector pTK27. (A) Structure of the pTK27 vector construct and its proviruses. Following transfection of the pTK27 vector, VSV-G envelope, and ΔNRF packaging construct into 293T cells, transcription of vector full-length RNA is initiated from the CMV promoter at the 5’ LTR. The vector RNA is packaged into infectious particles. In the process of reverse transcription the TRE from the 3’ LTR is transferred to the 5’ LTR. The presence of tTA in vector-transduced cells initiates full-length vector RNA that can be packaged into infectious particles. (B) FACS analysis of tTA-regulated expression of GFP. pTK27-transduced 293T cells were transfected with either tTA expression cassette (Clontech Tet-Off) or the control plasmid pBSK. 48 h posttransfection the cells were analyzed by FACS analysis for GFP expression. Nontransduced 293T cells served as negative control. (C) RNA analysis of tTA-dependent transcription of vector-length RNA. pTK27-transduced 293T cells were transfected as in (B). Total cellular RNA was extracted from each sample, ethidium bromide stained, and transferred to positively charged membrane. Transfer efficiency was determined by UV fluorescence (bottom). The positions of human 18S and 28S ribosomal RNAs are also marked. The blot was hybridized with 32P-labeled GFP probe. The expected migration of vector-length RNA is indicated. Molecular Therapy 2001 3, 97-104DOI: (10.1006/mthe.2000.0238) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 The effects of an internal CMV promoter on conditional SIN lentivirus vector. (A) Structure of the pTK27c vector, from which the GFP is expressed under the control of an internal CMV promoter. (B) Northern blot analysis of full-length vector RNA. pTK27c-transduced 293T cells were transfected with either a tTA expression cassette or the control plasmid, pBSK. Total cellular RNA was extracted 48 h posttransfection, stained with ethidium bromide, and transferred to positively charged membrane, which was hybridized with 32P-labeled probe containing the CMV promoter sequence. The expected migration of full-length vector RNA is indicated by an arrow. Efficiency of RNA transfer was determined by UV fluorescence (bottom). Arrows indicate the positions of 18S and 28S RNA. Molecular Therapy 2001 3, 97-104DOI: (10.1006/mthe.2000.0238) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Features of the cSIN lentivirus vector pTK136. (A) Structure of the lentivirus vectors: (top) pCL-CG, a non-SIN vector containing an intact U3 region in its 3’ LTR; (middle) pTK113, a simple SIN vector from which the HIV-1 promoter enhancer sequence including the TATA box was deleted from the 3’ LTR as indicated by Δ; (bottom) pTK136, a conditional SIN vector in which the transcriptional regulatory elements (excluding the TATA box) in the 3’ LTR were replace by the TRE. Regions containing the HIV-1 central purine tract and the woodchuck hepatitis virus posttranscriptional regulatory elements are indicated. (B) FACS analysis of GFP expression. 293T cells were transduced either by pTK113 or by pTK136 vector particles at m.o.i. of 0.3. 10 days postinfection cell samples were FACS analyzed for GFP expression. (C) Effects of HIV-1 packaging cassette on tTA regulation of full-length vector RNA transcription. PTK136-transduced 293T cells were transfected with the HIV-1 packaging construct ΔNRF either with a tTA expression cassette or with the pBSK control plasmid. Total cellular RNA was isolated 48 h posttransfection. RNA samples were ethidium bromide stained and transferred to positively charged membrane, which was hybridized with 32P-labeled probe containing the CMV promoter sequence. Efficiency of RNA transfer was determined by UV fluorescence (bottom). The expected migration of full-length vector RNA and size markers are indicated on the right. Molecular Therapy 2001 3, 97-104DOI: (10.1006/mthe.2000.0238) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 tTA-dependent mobilization of the conditional SIN lentivirus vectors. (A) High titers of the lentivirus vectors pTK136, pTK113, and CL-CG were generated by transient transfection and used to transduce HEK 293T cells at m.o.i. of 5. (B) Vector-transduced 293T cells were transfected with the VSV-G envelope and the ΔNRF packaging construct either with or without the tTA expression cassette. (C) Conditioned medium was collected 48 h posttransfection. (D) Mobilized vector titers were determined by scoring GFP expression following serial dilutions on HEK 293T cells. Molecular Therapy 2001 3, 97-104DOI: (10.1006/mthe.2000.0238) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Transduction of adult rat brain with the cSIN lentivirus vector pTK136. High-titer concentrated vector particle stocks (1 × 109 IU/ml) were generated by the stable lentivirus producer line SODk1cSCG. 1 μl of the concentrated cSIN vector stock was injected into striatum (A) and inferior colliculus (B) of adult rat brain. Expression of GFP in brain sections (40 μm thick) was determined by fluorescence microscopy 2 weeks postinjection. Molecular Therapy 2001 3, 97-104DOI: (10.1006/mthe.2000.0238) Copyright © 2001 American Society for Gene Therapy Terms and Conditions