Targeted insertion of a PGK-neo cassette does not disrupt the regulated expression of 2 neighboring murine β-major and β-minor globin genes.

(A) Real-time PCR assay for the wild-type mouse β-major genomic DNA. PCR product fluorescence intensity (ΔRn) is plotted on the y-axis versus PCR cycle number on the x-axis. Tenfold serial dilutions of pβ^WT, a plasmid encoding the wild-type β-major gene, were amplified using either the β^WT primer set (reactions 1-4) or β^6I primer set (reactions 5-8). Plotting the PCR cycle number at which the reaction entered exponential amplification, versus the logarithm of the amount of input DNA, generated a standard curve (not shown). (B) Ten nanograms plasmid DNA encoding the mutant β^6I (pβ^6I) or the wild-type β-major sequence (pβ^WT) was used for real-time PCR. Each plasmid sample or a no DNA control (No DNA) was amplified using either the β^WT primer set (reactions 1-3) or the β^6Iprimer set (reactions 4-6). (C) Real-time PCR analysis of bone marrow RNA. Bone marrow RNA was harvested from a heterozygous β^6I mouse (β^WT/β^6I) or a wild-type control mouse (β^WT/β^WT), reverse-transcribed to cDNA, and analyzed by real-time PCR using either the β^WT primer set (reactions 1-2) or β^6Iprimer set (reactions 3-4). A reaction amplifying the wild-type peripheral blood cDNA with the β^6I primer set was included as a negative control (reaction 5). (D) Semiquantitative RT-PCR analysis of peripheral blood RNA. Peripheral blood RNA was harvested from homozygous β^6I mice (β^6I/β^6I) or wild-type control mice (β^WT/β^WT), reverse-transcribed, and analyzed by PCR using a β-minor primer set or a β-actin gene primer set. Reactions were performed in the presence of α-³²P]dATP and stopped before amplifications reached a product plateau (not shown). Amplification products were resolved on 5% TBE-polyacrylamide gels and quantitated using a PhosphorImager. For each experiment, the relative β-minor mRNA expression was normalized for β-actin mRNA level and expressed as a percentage of wild-type expression. Shown are the mean values, along with standard deviations, from 3 separate experiments in which 2 samples were used for each genotype.