Twelve to sixteen weeks after CD34 + CD38 2 HSC engraftment, HIS mice were killed and single cell suspensions of splenocytes were prepared. Red cells lysis was performed in 1 ml of red cell lysis buffer (Sigma) for 10 min. Splenocytes were washed, resuspended in 600 ml of RLT lysis buffer (Qiagen) and homogenized by passing through a 21-gauge needle several times using RNase free syringes. RNA was prepared using RNeasy mini kits (Qiagen) according to manufactures instructions. BCR V H immunoscope was performed as previously described [17] . Briefly, cDNA was prepared and real-time PCR performed by combining primers for the different V H chains (V H 1-7) and specific fluorochrome-labeled probes against the different constant regions (C H m, C H a and C H c). An additional four PCR cycles 'run-off reactions' were then performed on the PCR products using fluorescent primers specific for the constant regions (Fcm, Fca and Fcc). Products were gel separated to determine CDR3 lengths. Analysis of six individual HIS-mice containing greater than 30% human chimerism in the spleen was performed. The number of human CD19 + B cells in chimeric spleens ranged from 5-12610 6 .