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

Figure 4

Discrimination between hemizygous and homozygous genotypes of transgenic mice by quantitative real-time PCR (a) or quantitative multiplex PCR of short fluorescent fragments (QMPSF) (b). (a) The amount of human β-globin allele was calculated using the comparative cycle threshold method [15, 33, 34] employing transgenic mouse TG24 as one copy control (hemizygous reference, grey). The black and white histograms represent hemizygous or homozygous transgenic (TG) mice, respectively. (b) Electropherograms obtained after denaturing polyacrylamide gel electrophoresis of amplification products of multiplex PCRs: primers employed (Table 1) were TransF[6FAM]-TransR and MuActF1[6FAM]-MuActR1, recognizing the transgene and the murine β-actin gene (Actβ), respectively; templates were genomic DNAs purified from a hemizygous (upper left panel) or a homozygous (upper right panel) mouse. Peaks generated by the molecular weight ladder (120, 150, 160, 180, 190, 200, 220 bp fragments) and by the amplification products (transgene and Actβ, indicated by the arrows) are reported in red and blue, respectively. Values obtained, as peak areas, transgene/Actβ ratio and fold of ratios calculated from homozygous and hemizygous animals are shown in the lower part of panel (b).

(a)

(b)