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3楼
发表于 2004-8-25 01:31
[B]Methods [/B]
Plasmids. Construction of pUC119CMVDHBV expression plasmid (1165A plasmid) has been described (15, 17). The 1165A mutation introduces a stop codon in the pre-S coding region of the envelope gene, causing a high level of viral DNA accumulation in the nucleus. The 1165A/DR1-13 plasmid containing a single-base change (C to G) on the plus strand at nucleotide position 2547 (18) was a generous gift from Dan Loeb (University of Wisconsin, Madison). The 1165A/DR1-13 plasmid is defective in plus-strand primer translocation, resulting in an 1:1 ratio of linear to circular DNA compared with an 1:10 ratio for the 1165A plasmid (15, 19, 20). To introduce a unique I-SceI restriction site into cells, we constructed a plasmid, pEGFP/I-SceI, that contained a single 18-bp I-SceI recognition sequence inserted into the enhanced green fluorescent protein (EGFP) gene, rendering the EGFP gene inactive, and a hygromycin-resistance gene to allow for selection of the integrated substrate. The starting plasmid for construction was pCM-VEGFP2 I-SceI(XhoI), a gift from Perry Kim (Queens University, Kingston, ON, Canada) and Jac Nickoloff (University of New Mexico). This plasmid contained two EGFP genes from pEGFP (Clontech) inserted into pCMV-Script (Stratagene). The cytomegalovirus (CMV)-EGFP gene contained an 18-bp I-SceI recognition sequence that had been inserted at an engineered XhoI site. Both EGFP genes were excised from pCM-VEGFP2 I-SceI(XhoI) by BamHI/HindIII digestion and subcloned into pcDNA3.1Hygro(–) (Invitrogen). The wild-type EGFP gene was deleted from this construct by digestion with HpaI/HindIII, filling in of the 5' overhang and ligating to create EGFP/I-SceI. Plasmid p1929, expressing EGFP, was obtained from Dan Loeb, and mRFP1, expressing monomeric red fluorescent protein, was a gift from Roger Tsien (University of California at San Diego, La Jolla).
Cell Culture and Transfections. Chicken hepatoma LMH cells were routinely maintained in DMEM/F-12 (1:1) supplemented with 10% FBS. To establish a stably integrated cell line containing an I-SceI substrate, pEGFP/I-SceI vector was linearized with SspI and transfected into LMH cells by electroporation. Briefly, 4 x 106 cells in 0.75 ml of PBS containing 1 µg of DNA were transferred to a cuvette with a 0.4-cm electrode gap and shocked with 300 V at 960 µF. Individual clones were selected and grown in medium containing 400 µg/ml hygromycin. Southern blot analysis was performed on isolated genomic DNA to identify a clone (LMH 3.2) containing a single-copy integrant of EGFP/I-SceI. Fifty micrograms of the I-SceI expression vector pCMV3xnlsI-SceI (21) plus 10 µg of 1165A/DR1-13 plasmid or a 1:1 mixture of 1165A and 1165A/DR1-13 were cotransfected into 4 x 106 LMH 3.2 cells by electroporation. Cells were plated and incubated for 3 days before the cell DNA was extracted for an assay of integrations.
In a reconstruction experiment to determine the relative efficiencies of transfection and cotransfection, EGFP- and mRFP1-expressing plasmids were cotransfected by electroporation at the same amounts as the experimental plasmids. The fraction of mRFP1-transfected (21%), EGFP-transfected (9%), and cotransfected (7%) cells was determined by fluorescence microscopy using red and green filters on an epifluorescent microscope (Nikon) by counting a minimum of 103 cells per transfection. These values were used to normalize the frequencies of imprecise NHEJ and DHBV integration to the number of cells transfected.
DNA Extraction. Cellular DNA was prepared from transfected cells by lysis of the cell layer of a 60-mm dish with 0.4 ml of SDS lysis buffer (10 mM Tris·HCl/10 mM Na-EDTA/0.5% SDS, pH 8.0) containing 0.5 mg/ml Pronase. After 1 h at 37°C, the lysate was extracted with an equal volume of phenol and recovered by ethanol precipitation. The nucleic acid pellet was dissolved in 0.1 ml of TE (10 mM Tris·HCl/1 mM Na-EDTA), adjusted to 1 µg/ml RNase A, and incubated for 10 min at 37°C. DNA was recovered by phenol extraction and ethanol precipitation. The final DNA concentration was determined by the optical density at 260 nm.
PCR Amplification. Individual left EGFP/DHBV junctions (see Fig. 1d) were detected by amplifying sequential dilutions of cellular DNA by nested PCR such that products were detected in only a fraction of the individual reactions. Nested PCR was performed in microplates by using 200 nM each of primers 1A and 1B (Table 1) and 25 units/ml AmpliTaq Gold (Applied Biosystems) in buffer containing 3 mM MgCl2 and 200 µM of each dNTP in a total volume of 10 µl. After an initial incubation at 95°C for 3 min, 40 cycles of PCR were performed by using a denaturation step of 95°C for 15 sec, annealing at 58°C for 15 sec, and extension at 72°C for 30 sec. Approximately 0.1 µl from each well was transferred to a replica microplate containing 10 µl of PCR mixture with 200 nM each of primers 2A and 2B and amplified for an additional 40 cycles. PCRs were electrophoresed through 1.3% agarose gels and stained with ethidium bromide. Right EGFP/DHBV junctions were amplified by using a similar nested-PCR strategy. Primer sets 3A and 3B followed by primers 4A and 4B were used to amplify individual right EGFP/DHBV junctions. To measure imprecise NHEJ of double-strand breaks (Fig. 1c), genomic DNA was digested for 12 h at 37°C with I-SceI (New England Biolabs) to enrich for cleavage-resistant sites. Nested PCR then was performed at limiting template dilutions using primer sets 1A and 3B followed by a second round of PCR using the primers 2A and 4B. The amplified products were digested with I-SceI to identify the nuclease-resistant products of imprecise NHEJ and detected by gel electrophoresis as described above. For quantification of replicative intermediates, known amounts of total DNA were amplified by real-time PCR using an iCycler (Bio-Rad). Forty cycles of amplification were performed as described above by using 1x iQ Sybr green supermix (Bio-Rad) containing 200 nM each of primers 5A and 1B (Table 1). |
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