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

Figure 10

Identification of the aberrant transcripts generated by the activation of the cryptic splicing site at position IVSI+13, in the presence of IVSI-6 mutation. (a) Schematic representation of the human β-globin pre-mRNA spanning from the first to the second exon (C); the creation of a new aberrant donor splicing site (GU) in the first intron, the IVSI-6 mutation site (star), the location of primers used for PCR (arrows), and the PCR product lengths are indicated. Normal splicing (A) and predicted aberrant splicing (B) are also represented. (b) RT-PCR reaction was performed by using RNA from healthy subject blood, from IVSI-6 homozygous patient blood and from transgenic mouse blood. The IVSI+13F forward primer and the HuBetaR reverse primer (Table 2), designed to amplify a fragment of 84 bp from human β-globin altered spliced transcripts or a fragment of 202 bp from human pre-mRNA (containing all the first human β-globin intron), were used. The products obtained from each sample, at 40 and 50 cycles of PCR reaction, were loaded on a 3% agarose gel. M, molecular weight ladder, pUC Mix Marker 8 (Fermentas), (−), negative control.

(a)

(b)