Generation of an AML1-ETO chimeric gene by homologous recombination. (A) Schematic of AML1 cDNA, partial murine AML1 genomic locus, and replacement vector containing a partial human AML1-ETO cDNA, polyadenylation signal (pA), and the positive selection neomycin resistance cassette (Neor) and negative selection diphtheria toxin-A cassette (DT-A). Arrows indicate the position of the AML1-ETO fusion and the transcriptional orientation of selection cassettes. The structure of the targeted allele and the chimeric AML1-ETO cDNA is shown, as are the primers used for RT-PCR amplification and detection of theAML1-ETO fusion transcript. Use of AML1 genomic probes A or B on Xba I–digested DNA allows resolution of wild-type and targeted alleles. (B) Southern analysis of control (CTR) and AML1-ETO knock-in (KI) ES cell clones. (C) RT-PCR analysis of CTR and KI ES cell clones. AML1-ETO mRNA was amplified using primers 1 and 2, and products were hybridized with the murine AML1-specific oligonucleotide 3. Amplification was also performed for HPRTmRNA as a control for the presence of amplifiable RNA.
