Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR

Rohr, Ulrich-Peter; Wulf, Marc Andre; Stahn, Susanne; Steidl, Ulrich; Haas, Rainer; Kronenwett, Ralf

doi:10.1016/s0166-0934(02)00138-6

Cited by 124 publications

(92 citation statements)

References 22 publications

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“…The observed titers in transducing units/ml (TU/ml) were roughly 1 log lower compared with the ones obtained as genomic RNA (RNAs/ml). This finding, being in agreement with earlier observations reported in the literature, [37][38][39] validates our real-time PCR method. The higher RNA titers are most likely due to the presence of defective interfering particles and do not appear to be due to plasmid DNA, as we conducted the PCR analysis of supernatants in the presence and absence of reverse transcription to exclude DNA background.…”

Section: Production Of Sfv Recombinant Particlessupporting

confidence: 93%

“…To remove contaminating DNA, that is plasmid DNA carried over from the production of vector, 38 viral RNA was treated with 30 U of RNase-free DNase I (Roche Diagnostics) at 37 1C for 30 min and heat-inactivated at 95 1C for 15 min, followed by quick cooling on ice. cDNA was prepared from 2 mg of total RNA in a 50 ml reaction mixture containing 1 mM Tris-HCl (pH 8.3), 5 mM KCl, 5.5 mM MgCl 2 , 2.5 mM random hexanucleotide primers, 0.5 mM of each dNTP, 40 U of RNase inhibitor and 125 U of MLV RT (Applied Biosystems, Foster City, CA, USA).…”

Section: Titration Of Recombinant Sfv Particles By Real Time Pcrmentioning

confidence: 99%

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Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

et al. 2008

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Section: Production Of Sfv Recombinant Particlessupporting

confidence: 93%

Section: Titration Of Recombinant Sfv Particles By Real Time Pcrmentioning

confidence: 99%

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

et al. 2008

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“…21 Briefly, 293T cells were cotransfected with 20 mg vector plasmid (pAAV-p53-AR6, 20 respectively, pAAV-GFP-AR6 22 ) and 20 mg helper plasmid (pDG 23 ) using calcium phosphate precipitation. Three days later, 293T cells were harvested and lysed by three freeze-thaw cycles following sonication.…”

Section: Production Of Recombinant Aav-2 Vector Stocksmentioning

confidence: 99%

The proteasome inhibitor bortezomib acts differently in combination with p53 gene transfer or cytotoxic chemotherapy on NSCLC cells

et al. 2007

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In this report, the effects of a combined treatment with the proteasome inhibitor bortezomib and either a recombinant adenoassociated virus type 2 (rAAV-2)-mediated p53 gene transfer or chemotherapeutic agents, docetaxel and pemetrexed, were tested on p53 positive and p53negative non-small cell lung cancer (NSCLC) cell lines. The combination of bortezomib and rAAV-p53 led to a significant synergistic inhibition of cell growth between 62-82% depending on the p53 status of the cell line and drug concentration. Surviving cells of the combined treatment showed a significant reduced ability to form colonies. Enhanced cell toxicity was associated with a 5.3-14.4-fold increase of the apoptotic rate and intracellular p53 level up to 50.4% following vectormediated p53 restoration and bortezomib treatment. In contrast, an antagonistic effect on tumor cell growth and colony formation was observed for the combination of bortezomib and docetaxel or pemetrexed as a reduction of cell growth between 31 and 48% was found in comparison to 50% using the single agents. Lower cytotoxic effects were associated with significantly reduced apoptosis and an increase of clonogenic growth. The observed antagonistic effects between bortezomib and docetaxel or pemetrexed might influence clinical trials using these compounds. Conversely, p53 restoration and bortezomib treatment led to enhanced, synergistic tumor cell toxicity.

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“…The general principles for making recombinant, replication-defective SV40 viral vectors have been already reported elsewhere. 23,24 SV40 stocks were titered by real-time quantitative Q-PCR as previously described, 25,26 with some modifications. Recombinant viral DNA was purified as previously described.…”

Section: Production Of Recombinant Sv40 Plasmidsmentioning

confidence: 99%

Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth

et al. 2006

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Pancreatic cancer is one of the most aggressive and devastating human malignancies. There is an urgent need for more effective therapy for patients with advanced disease. In this context, genetic therapy potentially represents a rational new approach to treating pancreatic cancer, which could provide an adjunct to conventional options. Because of the promise of recombinant SV40 vectors, we tested their ability to deliver a transgene, and to target a transcript, so as to inhibit pancreatic tumors growth in vivo. BxPC3 and Capan-1 cells were efficiently transduced using SV40 vectors without selection, as compared to synthetic vectors PEI. SV40 vectors were as efficient as adenoviral vectors, and provided long-term transgene expression. Next, we devised a SV40-derived, targeted gene therapy approach of pancreatic cancer, by combining hTR tumor-specific promoter with sst2 somatostatin receptor tumor-suppressor gene. In vitro cell proliferation was strongly impaired following administration of SV(hTR-sst2). SV40-derived sst2-mediated antiproliferative effect was dependent on the local production of somatostatin. In vivo, intratumoral gene transfer of sst2 using rSV40 vectors resulted in a marked inhibition of Capan-1 tumor progression, and proliferation. These results represent the initial steps toward a novel approach to the gene therapy of pancreatic cancer using SV40 as a vector.

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Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR

Cited by 124 publications

References 22 publications

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

Analysis of human immunodeficiency virus type 1 vector cis- and trans-acting elements production by means of Semliki Forest virus

The proteasome inhibitor bortezomib acts differently in combination with p53 gene transfer or cytotoxic chemotherapy on NSCLC cells

Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth

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