G3091

We determined the average human cell content of the chimeric liver using the TaqMan Copy Number Reference Assay (Applied Biosystems 4401613), which targets the single copy human RNase P gene. We took chimeric mouse liver that had been frozen in liquid nitrogen and powdered it using a mortar and pestle in liquid nitrogen. We isolated highly purified genomic DNA from aliquots of the powder by Proteinase K digestion, phenol and chloroform extraction, ethanol precipitation and spermine precipitation [23 (link)]. We constructed a standard curve for the TaqMan assay using known mixtures of human and mouse DNA (Promega G3041 and G3091).

Nuclear or whole lysate DNA was used from cultured cells or liver tissues. qPCR was performed with 2 uL of eluted DNA, in duplicate using Apex qPCR Green Master Mix (Genesee Scientific) and a CFX384 Touch Real-Time PCR Detection System (BioRad CFX Maestro (vl.1)) using the following cycling conditions: 95 °C for 15 min, 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 10 s and one cycle of 95 °C for 10 s and 65 °C for 1 min and 65–97 °C (5 °C s^–1). Standard curves for each primer set were generated using serially diluted plasmid (for gAAV) or Human and Mouse Genomic DNA (for host) (Promega G1521 and G3091, respectively) and used for quantification. CFX Maestro Software was used for data analysis. Sequence information for all qPCR primers is listed in Supplementary Data 1. Further analysis was performed in Microsoft Excel (v2111).