Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

Figure 6

Expression of human β-globin transcripts in the transgenic TG-β-IVSI-6 mouse model. (a) RT-PCR was performed with total RNA isolated from wild-type (lanes e and h), transgenic hemizygous (lanes c and f), and transgenic homozygous (lanes d and g) mice with primers HuBetaF1 and HuBetaR (Table 2) designed to specifically amplify a 153 bp fragment of human β-globin transcripts (lanes a–e) and primers MuBetaF and MuBetaR (lanes f–h) designed to specifically amplify a 147 bp fragment of mouse β-globin transcripts (black arrow). Genomic DNA and pCCL.β-globin.PGK.GFP.WPRE vector DNA were also used as control templates (lanes a and b, 283 bp, white arrow). M, molecular weight ladder, pUC Mix Marker 8 (Fermentas), (−), negative control for each primer pair. (b) SYBR Green real-time PCR was used to determine the relative expression of mouse α-globin, mouse β-globin, and human β-globin transcripts in wild-type mouse blood (left side of the panel) and in homozygous transgenic mouse blood (right side of the panel). (c) Relative expression levels of mouse β-globin (grey bars) and human β-globin (black bars) transcripts in transgenic mouse tissues using real-time RT-PCR. Mean ± SD values were determined for each fold difference; the relative proportions of β-globin/β-actin in each template were determined by using IQ5 software (Bio-Rad), employing the ΔΔCt method [15, 33, 34].