Highly sensitive quantification of human cells in chimeric NOD/SCID mice by real-time PCR.

We recently presented a real-time duplex PCR method to determine the amount of human cells in chimeric mice. Although this assay was suitable for the quantitative detection of 1% of human cells in the background of murine cells in tissue, blood and bone marrow of chimeric mice, there is legitimate interest in detecting even lower amounts of human cells in these animals reliably. Therefore, we developed a new assay that reliably detects as few as 5 human cells in 100,000 mouse cells (0.005%). The assay specifically amplifies a human and a murine endogeneous retroviral sequence in two separate reactions. Based on the same primers, the assay can be performed either with 5' nuclease probes (TaqMan assay) or with a pair of hybridization probes (LightCycler assay), enabling the use of various real-time PCR instruments. The use of immunodeficient mice in hematological studies has dramatically increased during the last years. One possible approach to quantify chimerism is based on specific detection of human DNA in chimeric tissue. Published PCR methods are either highly sensitive or allow reliable real-time quantification; however, sensitivity is usually reduced at the expense of quantification. For the investigation of putative bone marrow engrafting cell types, kinetic studies of early engraftment events or the colonization of individual mouse organs with human cells, the combination of both, high sensitivity and reliable quantification, is required. Therefore, we established a new real-time PCR based assay, which combines i) all advantages of real-time PCR, including quantification, reduced contamination risk and specificity, with ii) an extremely high sensitivity. The assay is based on separate amplification of the human specific endogenous retroviral sequence ERV-3 and the mouse specific endogenous retroviral sequence ERV-L pol. For the detection of human ERV-3 a recently published TaqMan assay was modified and suitable hybridization probes were designed, whereas the mouse ERV-L assay was a completely new development. Both assays were optimized to be run under identical reaction conditions, permitting a simultaneous analysis of human and murine cells. TaqMan PCR was performed in a Perkin Elmer 7700 Sequence Detection System in 96-well microtiter plates in a final volume of 25 μL including 2.5 μL 10x PCR reaction buffer, 4.5 mM MgCl2, 1.0 mM dNTP, 1 U Platinum Taq DNA polymerase (all Invitrogen, Karlsruhe, Germany), 5 pmol of each primer, 3 pmol 5’ nuclease probe and 1 μM ROX (6-carboxy-X-rhodamine). 2 μL template DNA were amplified starting with 3 min at 94°C, followed by 45 cycles of 94°C for 30 s and at 58°C for 30 s. LightCycler PCR (Roche Molecular Diagnostics, Mannheim) was performed in a final reaction volume of 20 μL including 2 μL LightCycler-Fast Start DNA Master Hybridisation Probes mix (Roche, Mannheim, Germany), 5 mM Mg, 6 pmol of each primer, 2 pmol of each hybridization probe and 2 μL template DNA. Cycling conditions were 10 min at 95°C followed by 45 cycles of 8 s at 95°C, 8 s at 51°C and 8 s at 72°C. Table 1. shows primers and detection probes used for TaqMan and LightCycler PCR. To prove the sensitivity of the assay, human MCF-7 cells were diluted stepwise in murine P388 cells from 100% human cells to 0.005% human cells. DNA preparation was performed as described previously. Both assays were specific for each species. As shown