HIV 1 and lentiviral vector preparation The preparation and

HIV 1 and lentiviral vector preparation The preparation and titration of replication proficient and VSVG pseudotyped viruses are described elsewhere. The lentiviral vectors were prepared utilizing pLenti6 EGFP Hedgehog agonist and the ViraPower Lentiviral Packaging Mix based on the company s method. Viral supernatants were centrifuged at 120 g for 5 min, filtered via a 0. 2 um filter, and saved at 80 C. To exchange the medium for DMEM supplemented with 0. One of the FBS, the infections were ultracentrifuged at 86,000 g for 1 h.. Quantitative PCR of provirus DNA For your quantification of early RT, late RT, 2 LTR circle, and built-in DNA, qPCR was performed as described elsewhere. Shortly, cells were collected at 48 hpi, and genomic DNA was prepared by QuickGene. For neuroendocrine system the quantification of early RT, late RT, and 2 LTR range products and services, the primers and probe sets M667/ AA55/R U5, M667/M661/R U5, and MH535/2 LTR AS/ NL4 3 U3 were used, respectively. TaqMan Universal PCR Master Mix with UNG and ABI7000 were used according to the maker s directions. For Alu PCR, the primer and probe sets first Alu F/ first Alu R/first gag R and second tag R/2 LTR S/probe 2 were used for the first and second rounds of qPCR, respectively. The amplification conditions for the first round of PCR, using AmpliTaq Gold 360 Master Mix, were as follows: 95 C for 10 min, accompanied by 12 cycles at 95 C for 15 s, 60 C for 30 s, and 72 C for 10 min. The second round of qPCR was done using TaqMan Universal PCR Master Mix based on the manufacturer s instructions. To generate a typical curve for Alu PCR, HEK293T cells were infected with VSVG pseudotyped NL Luc E Kiminas disease, then harvested at 30 d postinfection, and genomic DNA was prepared. For the quantification of B globin DNA copy figures, the primer set globin F/globin R was used with SYBR Pre-mix ExTaq. EMD?121974 Sequence information for primers and probes is listed in Additional report 1: Dining table S2. . Cleavage of I SceI and I PpoI sites Ad I SceI and Ad LacZ were prepared as described previously. PMA treated THP 1 cells were contaminated with Ad I SceI or Ad LacZ at 1 h post HIV 1 infection for 1 h at a multiplicity of infection of 100. In Figure 1E 1H, HT1080 cells were transfected with plasmid DNA that encoded a chmeric protein of estrogen receptor I PpoI, and then 4 hydroxytamoxifen was added to induce DSB and trigger the endonuclease. The pAxCALNLwtit2 cosmid vector harboring I PpoI cDNA was digested with BspT104I and transduced in to HEK293 cells to make Ad I PpoI. The adenoviral vector encoding Cre recombinase, AxCANCre, was coinfected with Ad I PpoI at an MOI of 30 to eliminate the floxed stuffer involving the CAG promoter and I PpoI cDNA. Quantification of the I SceI site specific viral integration PMA addressed THP 1 cells were co attacked with WT disease and Ad I SceI or Ad LacZ, and then extracted genomic DNA was afflicted by I SceI qPCR analysis.

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