Comparison of the level of human β globin gene expression in YAC transgenic lines. (A) Autoradiograph of products of RT-PCR. Reverse transcription of 1 to 500 ng total blood RNA from line A20.1 (first 5 lanes), A20.1 transgenic RNA sample with trace genomic DNA present (F1), and RNA from a nontransgenic animal (F1-), followed by PCR amplification with human β globin primers. (B) Ethidium-stained acrylamide gel showing products of RT-PCR of total blood RNA using murine β globin primers, followed by digestion with Bstx1: line A20.1 on C57Bl6 (HbbS) genetic background (lane 1), A20.1 in mice heterozygous for the diffuse and single murine β globin alleles (lane 2), A20.1 on FVB/NJ (HbbD) background (lane 3), and nontransgenic C57Bl6 (lane 4) and FVB/NJ (lane 5) RNA. M, loaded with the φx174/HaeIII marker ladder. (C) Autoradiographs of RT-PCR products resolved by electrophoresis in 5% acrylamide gels. Samples from β globin YAC transgenic lines are indicated above each set of 4 lanes. For each line, 10-fold serial dilutions of total blood RNA were made, and 1 μg (lanes 1, 5, 9, and 13), 100 ng (lanes 2, 6, 10, and 14), 10 ng (lanes 3, 7, 11, and 15), or 1 ng (lanes 4, 8, 12, and 16) RNA was included in the RT reaction. PCR was performed in the presence of both human and mouse β globin primers. Lane F1, RT-PCR of 100 ng total blood RNA from a nontransgenic animal showing amplification of murine β globin RNA. Lane M, φx174/HaeIII marker ladder. The % human β globin/mouse β globin corrected for the number of human and mouse loci and standard deviations calculated for each line are indicated.
