Lentivirus Titering

HIV-PSI F 5 -ACCTGAAAGCGAAAGGGAAAC
HIV-PSI R 5 -CGCACCCATCTCTCTCCTTCT
HIV-PSI-FAM 5 -6-FAM-AGCTCTCTC-ZEN-GACGCAGGACTCGGC-Iowa Black FQ

SDC4 F 5 -CAGGGTCTGGGAGCCAAGT
SDC4 R 5 -GCACAGTGCTGGACATTGACA
SDC4-HEX 5 -HEX-CCCACCGAACCCAAGAAACTAGAGGAGAAT-Iowa Black FQ
uc483 F 5 -GCATGCTTCATTAACAGTGACC
uc483 R 5 -TTTAAAATCTGAATGCATGATAAGAATGG

uc483-HEX 5 -HEX-AGATCCCCAGCTCATCCGTGATTG-Iowa Black FQ

A standard curve was prepared from samples of HT29 DNA mixed with DNA from a HT29 clone that has 2 copies/cell of a
lentiviral vector.

To obviate DNA concentration determination and normalization, a multiplex probe-based real-time PCR reaction was used combining primers and probe to detect a conserved LV sequence (HIV-1 psi region) with primers and probe targeted to the autosomal gene syndecan 4 (SDC4, De Preter et al., 2002) for normalization (Table 1). The SDC4 internal control allows the cycles to threshold (Ct) value of psi to be normalized to that of the endogenous control, which is reflective of the number of cell equivalents of DNA present in the reaction. Therefore, the same volume of DNA can be added to each reaction even though the concentrations will be somewhat different. Substitution of the SDC4 primers and probe with those directed to the ultra-conserved region uc483 (200 nM each primer, 100 nM probe) allows normalization of genomic DNA from mouse as well as human cells and many other vertebrate cells, if there is a need to determine titer in cells of another species (Bejerano et al., 2004). However, the uc483 primers and probe must be used in parallel reactions rather
than in multiplex

Real-time PCR was performed using ABI TaqMan Universal PCR Master Mix and an ABI 7500 Fast Real-Time PCR System. A total volume of 25 ␮L was used for reactions with 400 nM each for SMPU primers, 50 nM each for SDC4 primers, 50 nM each probe, and 1 ␮L of genomic DNA template (50–300 ng). Cycling
conditions were as recommended by the manufacturer, and the ‘fast’ option of the system was not used

In order to interpret the data, the Ct was determined for each well ( Ct = Ctpsi − CtSDC4 ). A standard curve was plotted as a log2 (copy number) vs. Ct. The standard curve DNA had copy numbers of 2, 0.2, 0.02, 0.002, and 0.0002, corresponding to 100 through 10−4 dilutions. A linear equation was obtained
for a best-fit line of the standard curve. The Ct values for each experimental sample were put into the equation to obtain the log2 (copy number).

copy number = 2log2 (copy number) and titer was determined with the following equation:
Titer (TU/mL) = (cell count at time of transduction) × (copy number)(dilution factor)