Development of an HIV-Based cDNA expression cloning system Marc van Maanen, Jennie K Tidwell, Lawrence A Donehower, Richard E Sutton  Molecular Therapy  Volume 8, Issue 1, Pages 167-173 (July 2003) DOI: 10.1016/S1525-0016(03)00133-3 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Structures of HIV-based vectors for cDNA library transfer. (A) Schematic structure of provirus NL4-3, with deletions indicated by delimited bars. R denotes the RRE, Ψ is the packaging signal. (B) Scale schematic showing structure of the cDNA expression vector pHIV-ΔNS-DNA-IRES-Blasti. Arrows (not to scale) indicate flanking PCR primers. (C) Vector DNA sequence at the multiple cloning site (MCS) of pHIV-ΔNS-DNA-IRES-Blasti. Also shown are U and L primers for insert rescue, the positions of the unique restriction enzyme sites, and the position of the mutated Nef start codon. Molecular Therapy 2003 8, 167-173DOI: (10.1016/S1525-0016(03)00133-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Schematic for proof-of-concept expression cloning experiments using HIV-based cDNA libraries. In the first step (top left) 293T cells are transfected with relevant plasmids and after 3 days collected vector supernatant is used to transduce targets (top right). After 2 more days, transduced cells are preselected using antibiotic (bottom right), and surviving cells are then subjected to the genetic selection of interest (bottom left). Molecular Therapy 2003 8, 167-173DOI: (10.1016/S1525-0016(03)00133-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Recovery of cDNA inserts using vector-specific primers. Genomic DNA was isolated from HATR cell clones following selection for either (A) TK or (B) HPRT expression by growth of cDNA library-transduced cells in HAT-containing medium. (A) Lanes: M, 1 kb Plus DNA ladder (Invitrogen, CA); (−), water, negative control; A1–A6, individual HATR cell clones from functional TK screen, using HOS TK− cells transduced with the HFF-derived cDNA library. (B) Lanes: M, 1 kb Plus DNA ladder; (−), water, negative control; B1–B6, individual HATR cell clones following screen for HPRT function, using HPRT− cells transduced with the PM1-derived cDNA library. Molecular Therapy 2003 8, 167-173DOI: (10.1016/S1525-0016(03)00133-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Functional cDNA library screening in primary, mitotically inactive HFFs. (A) Photomicrograph of senescent HFFs at PD 69 stained with crystal violet. Scale bar, 100 μm. (B) Photomicrograph of crystal violet-stained senescent HFFs (PD 69), 14 days after transduction with an HIV cDNA expression vector containing the SV TAg cDNA. (C) Genomic DNA was isolated from four rapidly proliferating cell clones following transduction of senescent HFFs with vector generated by cotransfection of HIV-PM1 cDNA library and HIV-TAg-IRES Blasti plasmid (mass ratio of 25,000:1). Lanes: M, 1 kb Plus DNA ladder; C1–C4, individual cell clones recovered from SV TAg screen; (−), water, negative control. (D) Autoradiograph showing results of Southern blot analysis performed on the gel shown in C. PCR products were transferred to nylon membrane and probed using radiolabeled SV TAg cDNA. Membranes were washed for 60 min in 0.1× SSC/0.1% SDS and exposed to film for 2 min. Lanes are as described for C. Molecular Therapy 2003 8, 167-173DOI: (10.1016/S1525-0016(03)00133-3) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions