Integrated lentivirus copy number quantification

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LVpro Provirus qPCR Quantitation Kit workflow

LVpro Provirus qPCR Quantitation Kit workflow. A calibration curve is created using the Provirus Control Template, which contains one copy of each of the lentiviral sequence and a part of the human IFNγ sequence. The average number of copies of the provirus per cell is calculated by quantifying the copy number of lentiviral and human IFNγ in the genomic DNA of the target sample.

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Close correlation of provirus copy numbers with WGS

Close correlation of provirus copy numbers with WGS using LVpro Provirus qPCR Quantitation Kit. Autosomal diploid cells transduced with a lentiviral vector were cloned, and the proviral copy number was determined by whole-genome sequencing (WGS) from the extracted genomic DNA. In addition, 100 ng of genomic DNA extracted from each clone was used to measure the number of proviral copies by the LVpro Provirus qPCR Quantitation Kit or other companies' products. The results showed that the number of proviral copies determined by the LVpro Provirus qPCR Quantitation Kit and the number of proviral copies obtained by WGS analysis were almost equivalent, suggesting that the quantification of this product is highly accurate.

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High correlation between MOI and proviral copy number

High correlation between MOI and proviral copy number. (Top) Lentiviral vectors containing the ZsGreen1 gene were phased and infected with HT1080 cells with various multiplicity of infection (MOIs). Three days after infection, the cells were treated with DNase I and then genomic DNA was extracted using Nucleospin Tissue. (Bottom) Lentiviral vectors carrying the CD19-CAR gene were phased and infected with SUP-T1 cells with various MOIs. Three days after infection, the cells were treated with DNase I, and then genomic DNA was extracted using Nucleospin Tissue. Real-time PCR was performed using 100 ng of genomic DNA for each sample. The correlation between the average number of proviral copies per cell and MOI was confirmed. The data present the average of the three independent trials. As a result, the combination of different cell lines and different carrier genes showed that the MOI and proviral copy number R² exhibited a high correlation with values above 0.98.

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Measuring lentiviral copy number in target cells

Measuring lentiviral copy number in target cells. Provirus content is determined using a rapid qPCR method.

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Lentiviral provirus copy number affects transgene expression levels in transduced cells

Lentiviral provirus copy number affects transgene expression levels in transduced cells. HT1080 cells were infected at either high or low MOI using an AcGFP1 lentiviral expression vector. 72 hr after infection, the cells were subjected to puromycin drug selection. One week later, stably transduced clones were selected for expansion and analysis. AcGFP1 expression levels were determined using flow cytometry, and provirus content in genomic DNA was determined using the Lenti-X Provirus Quantitation Kit. The figure represents data pooled from two groups having either low copy number (>3; n = 8) or high copy number (>9; n = 7). Higher provirus contents were associated with higher expression levels.

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Equivalent virus amounts in different cell types result in different provirus (integrated lentivirus) copy numbers

Equivalent virus amounts in different cell types results in different provirus (integrated lentivirus) copy numbers. Equivalent amounts of a single lentiviral stock (100 µl) were used to infect either HT1080, HeLa, and HEK 293 cells. 72 hr after infection, the cells were subjected to drug selection with puromycin. One week later, the provirus copy numbers of the stably transduced cell populations were determined using the Lenti-X Provirus Quantitation Kit.

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631239: Lenti-X Provirus Quantitation Kit