Murine Leukemias with Retroviral Insertions at Lmo2 Are Predictive of the Leukemias Induced in SCID-X1 Patients Following Retroviral Gene Therapy
Figure 2
Tumor 7107 has two independent insertions in Il2rg and Irs2 genes.
A) LM-PCR identified two independent insertions in tumor 7107, 5′ of the eight coding exons of the Il2rg gene. The two viral chromosomal junctions were independently cloned and sequenced. The insertions were 396 bp apart and should be present in the same clone if they occurred on the same chromosome. Partial sequence for clone 23-10 shows the region of identity with clone 23-1 (green). For clone 23-1, we found viral sequence 5′ of this region instead of chromosome X genomic sequence. B) Two insertions in the Irs2 gene were also isolated from the same tumor. Primers were designed for one allelic insertion for Irs2 and Il2rg and real time PCR performed on the genomic DNA. Primers for a chromosome X sequence were used as reference and show a copy number of 1 for this male mouse. As shown, each insertion comprises about 50% of total tumor genomic DNA. We ruled out polyploidy for Irs2 and Il2rg genes (see Figure S4). C) Panel shows a heat map of gene expression data using the mouse Affymetrix array. The expression of genes near high frequency insertions cloned from 7107 is shown with respect to normal thymus. Log2 intensity scale is shown under the heat map.