Figure 8.

RT-PCR analysis of transgene and endogenous cytokine receptor gene expression in primitive hematopoietic progenitors. RNA was prepared from 10 Lin⁻Sca-1⁺c-kit⁺CD34⁻ cells obtained by clone sorting of BM cells of the chimeric receptor transgenic mice with 10 μg of polyinosinic acid as an RNA carrier. Using one quadrant of the RNA, cDNA was synthesized. PCR was run for 40 cycles. After removal of the first PCR primers, 0.1 of the first PCR product was used for the second nested PCR. The second PCR was run for 40 cycles. The expected sizes of the final PCR products were hGM-CSFRα, 598 bp; hGM-CSFRβ, 554 bp; mgp130 (extracellular domain), 464 bp; mIL-6Rα, 556 bp; and mIL-11Rα, 429 bp. The suitability of the primers for mgp130, mIL-6Rα, and mIL-11Rα was confirmed by PCR using cDNA of 10 BM cells of 5-FU-treated C57BL/6 mice as a template. These data are from a single experiment, and similar results were obtained in two additional experiments.