Kubo et al. 10.1073/pnas.0601954103. |
Supporting Materials and Methods
Supporting Materials and Methods
Cells.
The human embryonic kidney cell lines 293 (1), 293T (2), 293 FLPe (3), and the human glioma cell line Gli36 (4) were cultured in DMEM supplemented with 10% FCS (Omega) in a humidified incubator at 37°C with 5% CO2. Hep3B human hepatocellular carcinoma cells were cultured in Eagle’s minimal essential medium (MEM) supplemented with 10% FCS/1 mM sodium pyruvate, and nonessential amino acids. Primary human dermal fibroblasts (NHDF; CC-2509), hepatocytes (CC-2591), and their specific media were purchased from Clonetics.Adenovirus-Retrotransposon Hybrid
(A/RT) Vector Plasmid Construction. The retransposition-competent (RC)-long interspersed element-1 (L1) retrotransposon-reporter cassette system consists of a L1RP retrotransposon sequence, a phosphoglycerate kinase-1 (pgk) promoter, and a EGFP reporter gene cassette (5, 6). First, a marker gene cassette, containing the b-gal gene driven by the simian virus (SV) 40 promoter, was subcloned into the pSTK68 adenovirus backbone (7) to generate the intermediate construct pSTKb-gal. Next, the L1RP retrotransposon-EGFP reporter gene cassette was inserted into pSTKb-gal. The resultant plasmid, pA/RT-pgk-L1RP-EGFP, thus contains a Hhelper-dependent adenovirus construct that encodes two independent cassettes, SV40-b-gal and pgk-L1RP-EGFP. A detailed description of this subcloning is available upon request.Lentiviral Vector Construction and Production.
The lentiviral vector construct, psinSKcmv-EGFP, was generated by insertion of a central polypurine tract and termination sequences into pRRL-sin.hCMV-EGFP-pre upstream of the EGFP as described by Follenzi et al. (8). Lentivirus preparations were produced from 293T cells by standard 4-plasmid calcium phosphate cotransfection methods by using pMDLg/p (encoding gag-pol), pRSV-Rev (encoding rev), and pMD.G (encoding VSV-G env) (9).Retroviral Vector Construction and Production.
The retroviral vector construct pACEemd has been described in ref. 10 and encodes a fully replication-competent amphotropic Moloney murine leukemia virus (MLV) vector, in which the 5' LTR U3 region has been replaced with the CMV promoter, and an internal ribosome entry site- EGFP marker gene cassette has been inserted between the amphotropic env gene and a 3' UTR. Retrovirus preparations were produced by transient transfection of 293T cells with pACEemd (10). For in vitro transduction experiments, 4 mg/ml polybrene (Sigma) was added to the medium at the time of infection.Plasmid Transfection Experiments.
For direct plasmid transfection experiments, 1 × 106 cells in a six-well plate were transfected with 4 mg of pA/RT-pgk-L1RP-EGFP or control plasmids, pEGFP-N1 (Clontech), by Lipofectamine 2000, passaged at a ratio of 1:20 every week. EGFP expression was examined by an EPICS XL-MCL flow cytometer (FACS; Beckman-Coulter).Production of A/RT Helper-Dependent Adenovirus.
The A/RT-pgk-L1RP-EGFP virus was prepared by using the FLPe/FRT helper virus system, as described in ref. 3. Briefly, linearized vector constructs were transfected into 293FLPe cells in the presence of FL helper adenovirus, propagated serially, and purified by CsCl ultracentrifugation. The b-gal titer of the A/RT virus in transducing units per ml was determined by infection of 293 cells at various dilutions, followed by X-Gal staining and quantitation of positive cells. Helper virus contamination levels in the vector stock were determined by Southern blot hybridization by using a probe for the adenoviral packaging signal (7).Infection Experiments with A/RT Hybrid Virus and Analysis of Retrotransposition.
To confirm retrotransposition in cells after viral infection, 2 × 105 cells in a 12-well plate were infected with the A/RT-pgk-L1RP-EGFP virus at various multiplicities of infection (mois) in quadruplicate. Seven days later, expression of EGFP was examined by FACS analysis, and b-gal was examined by staining. To confirm retrotransposition in primary human cells after viral infection, 2 × 105 hepatocytes and dermal fibroblasts in 12-well plates were infected with A/RT virus at various amounts. Seven days later, expression of EGFP and b-gal was examined as above.To investigate the kinetics of viral transduction, 5 × 105 Gli36 cells were infected with A/RT virus at a moi of 10 in triplicate. The cells were collected every day and analyzed by FACS. For longer time points beyond 1 week after tinfection, 1 × 105 cells in 12-well plates were infected with A/RT virus at various mois in quadruplicate. The cells were split at a ratio of 1:20 on a 10-cm dish once a week, and expression of EGFP and b-gal was examined. Over the course of several passages, genomic DNA was extracted from a portion of the cells and analyzed for retrotransposon integration by PCR as described in refs. 5 and 6.
To examine whether retrotransposition after A/RT virus infection is inhibited by reverse transcriptase inhibitors, 4 × 105 cells were infected with either A/RT virus at a moi of 10 or control lentivirus vector (sinSKcmv-EGFP) and incubated in growth medium in the presence or absence of 3'-azido-3'-deoxythymidine (Zidovudine, AZT; 5 mM) (Sigma), and FACS analysis was performed as described above.
Isolation of Postintegration Sites via Inverse PCR.
Gli36 cell DNA (4 mg) derived from individual A/RT-pgk-L1RP-EGFP-infected EGFP-positive cells was digested with XbaI or SspI (NEB). The resultant DNA was subjected to overnight self-ligation (11, 12). The products then were phenol extracted, precipitated with ethanol, and subjected to nested-PCR amplification with two different sets of primers for the EGFP expression cassette. The resultant PCR fragments were cloned into TOPO-XL-PCR (Invitrogen). The primer sequences used for inverse PCR are available upon request.Immunocytochemistry for BrdU Incorporation and EGFP Expression.
Gli36 cells (1 × 105) on day 1 after plating were infected with A/RT virus at a moi of 10 and incubated in the presence of 10 mM BrdU (Sigma). Five days later, cells were stained for BrdU incorporation by BrdU in situ detection kit (BD Biosciences) and DAB substrate kit (Vector Laboratories). After blocking endogenous peroxidase activity by incubating cells with in 0.3% H2O2/PBS, cytoplasmic EGFP was detected by incubating cells then with a rabbit polyclonal GFP-specific antibody (ab290, Abcam; diluted 1:200), visualized by using Vectastain ABC kit (Vector Laboratories) and Vector NovaRED substrate kit (Vector Laboratories).Cell Cycle Analysis.
G1/S-arrested Gli36 cells were prepared by seeding 2 × 105 cells per well in a six-well tray 2 days before infection and adding aphidicolin (10 mg/ml) in the culture medium with reduced serum (5%) 24 h before and after infection. G0-phase cells were prepared by maintaining the cells at confluency in the culture medium with reduced serum (2%) for 1 week. In the experiment shown in Fig. 4a, seven sets of these G1/S-arrested Gli36 cells were infected with A/RT virus at a moi of 10. On the following day, three sets of G1/S-arrested cells were analyzed for cell cycling by FACS analysis after propidium iodide staining and immunoblot analysis for cell cycle-related proteins Ki67 and cyclin D1, as well as for both EGFP and b-gal expression as described above. One of the sets then was split at a ratio of 1:10 (growing) into three new wells. At 5 days after infection, these cells were analyzed for cell cycle status and expression of EGFP and b-gal. In other experiments, the G1/S-arrested and G0 cells prepared as above were infected with a replication-competent MLV retrovirus (ACEemd, MLV) (10), an HIV-based self-inactivating lentiviral vector (sinSKcmv-EGFP, HIV), or the A/RT-pgk-L1RP-EGFP virus. One set of G0 phase cells transduced with A/RT-pgk-L1RP-EGFP virus, which did not show retrotransposition, was replated at a ratio of 1:20 to induce cell cycling and analyzed for EGFP expression at 7 days after infection.Statistical Analysis.
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