Volume 8, Issue 1, Pages 158-166 (July 2003) A simple and efficient method for constructing an adenoviral cDNA expression library  Kazuteru Hatanaka, Shumpei Ohnami, Kimiko Yoshida, Yoshiaki Miura, Kazuhiko Aoyagi, Hiroki Sasaki, Masahiro Asaka, Masaaki Terada, Teruhiko Yoshida, Kazunori Aoki  Molecular Therapy  Volume 8, Issue 1, Pages 158-166 (July 2003) DOI: 10.1016/S1525-0016(03)00138-2 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Construction of an adenovirus cDNA expression library. The linearized shuttle plasmid cDNA library is mixed with left-end-digested adenoviral DNA-TPC, and Cre recombinase produces the full-length recombinant adenoviral DNA library in vitro. The Cre-treated DNA library is transfected into high-efficiency virus-producing 293 cells, and a recombinant adenovirus cDNA library is produced. Molecular Therapy 2003 8, 158-166DOI: (10.1016/S1525-0016(03)00138-2) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Transfection method for 293 cells. 2–10 μg of adenoviral genomic DNA containing CMV-AP was transfected into 293 cells in a six-well plate, and then the expression of the AP gene was evaluated. (a) Calcium phosphate (CellPhect Transfection Kit) using 10 μg of DNA. (b) Lipofection (Lipofectamine Plus reagent) using 6 μg of DNA. (c) Radiofrequency electroporation [31] (Gene Pulser II RF module) using 10 μg of DNA. (d) Polyethylenimine [32] using 6 μg of DNA. Molecular Therapy 2003 8, 158-166DOI: (10.1016/S1525-0016(03)00138-2) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Enhancement of adenovirus production by terminal protein. (A) Experimental design of dilution experiment. The pAdCMV-EGFP were mixed with pAdCMV-AP or a shuttle plasmid library at various ratios, recombined with cS360loxP or DNA-TPC by Cre in vitro, and then transfected into parental 293 cells and their high-efficiency virus-producing clones. After 6–9 days, 7% of viral lysates was infected into A549 cells to detect an EGFP-expressing adenovirus. (B) EGFP expression in A549 cells at 48 h postinfection (×100 original magnification). Various ratios of pAdCMV-EGFP and pAdCMV-AP were recombined with left-end-digested DNA-TPC and then transfected into 293 clones. Titers of each adenoviral lysate, which were determined by the TCID50 assay [33], were 8.2 × 108 to 5.9 × 109 PFU/ml. (C) Southern blot analysis of adenoviral library DNA. Various ratios of pAdCMV-EGFP and shuttle plasmid library were recombined with left-end-digested DNA-TPC and then transfected into clone 38. Two micrograms of the EcoRI- (lanes 2 and 3) or XbaI-digested cell lysates (lanes 5 and 6) were analyzed by Southern blot hybridization with a 32P-labeled 0–1 mu + CMV promoter (left end) probe (lanes 1–3) or an EGFP probe (lanes 4–6). To examine the amount of ADVCMV-EGFP DNA within the total adenovirus DNA, the exposure time was adjusted to show the same band intensity in the control plasmid lane (10 ng of pAdCMV-EGFP) (lanes 1 and 4) of each film. The left side arrow indicates the band of the left end from the total adenoviral library DNA at 30 (lane 2) and 300 (lane 3) dilutions. The right side arrow indicates the band of the left end and the EGFP gene from ADVCMV-EGFP at 30 (lane 5) and 300 (lane 6) dilutions. The ratio of ADVCMV-EGFP DNA within the total adenoviral DNA, which was determined by densitometry, was approximately 1:30 (lane 5:lane 2) and 1:300 (lane 6:lane 3). (D) PCR analysis of plasmid library and adenoviral library DNA. PCR amplification of six endogenous genes was carried out in DNA of the shuttle plasmid library and adenoviral library using specific primers. PCR products were confirmed by sequencing. Lane 1: RT-PCR using total RNA extracted from human T cells. Lane 2: shuttle plasmid library derived from human T cells. Lane 3: adenoviral library DNA converted from shuttle plasmid library. Lane 4: no DNA. (E) PCR analysis of adenoviral library DNA during multiple-round amplifications of viruses. Adenoviral library derived from human T cells was infected into 293 cells, and then the crude viral lysate was harvested and used to infect 293 cells. This process was repeated. PCR amplification of six endogenous genes was carried out in DNA of adenoviral libraries during three rounds of amplification using specific primers. Lane 1: adenovirus library DNA after first round of amplification. Lane 2: second round of amplification. Lane 3: third round of amplification. Molecular Therapy 2003 8, 158-166DOI: (10.1016/S1525-0016(03)00138-2) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Screening of the viral cDNA library. (A) Strategy for identification of CD2 gene with adenoviral cDNA library. In the first-round screening, the shuttle plasmid library was divided into 96 cDNA pools (about 100 colonies/pool). Each pool was recombined with DNA-TPC by Cre in vitro and transfected into clone 38 cells in a 96-well collagen-coated microplate. After 8 days, cell lysate-infected AsPC-1 cells were examined by immunocytochemistry. In the second-round screening, each colony from a CD2-positive pool was again examined by the same procedure. (B) Southern blot analysis to estimate the frequency of CD2 plasmid. Various amounts of pAdCMV-CD2 and 1 μg of shuttle plasmid cDNA library constructed from human T cells were analyzed by Southern blot hybridization with a 32P-labeled CD2 cDNA probe. Lanes 1–7: pAdCMV-CD2 [lane 1, 1 μg; lane 2, 100 ng (1/10 of lane 1); lane 3, 10 ng (1/100); lane 4, 1 ng (1/1000); lane 5, 200 pg (1/5000); lane 6, 100 pg (1/10,000); lane 7, 20 pg (1/50,000)]. Lane 8: T cell shuttle plasmid library (1 μg). CD2 clone in human T cell cDNA library was estimated at less than 1/3000 by densitometry. (C) Immunocytochemistry for CD2. Library-infected AsPC-1 cells were stained with antibody to human CD2. Upper left: AsPC-1 cells showing positive staining. Upper right: AsPC-1 cells showing negative staining. Lower left: positive control (ADVCMV-CD2-infected AsPC-1 cells). Lower right: negative control (ADVCMV-EGFP-infected AsPC-1 cells). (D) PCR analysis to identify CD2 cDNA. Positive pools were analyzed by PCR using the same CD2-specific primers as in Fig. 3D. Lane 1: 500 bp ladder marker. Lane 2: DNA from a shuttle plasmid pool showing positive staining (from C, upper left). Lane 3: DNA from a shuttle plasmid pool showing negative staining (from C, upper right). Molecular Therapy 2003 8, 158-166DOI: (10.1016/S1525-0016(03)00138-2) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions