Human β-globin transgene integrity and copy number analysis in stably transfected MEL cell clones.

<p>(A) Representative Southern blot analysis of DNA from MEL cell clones stably transfected with wild-type (βWT) or nonsense-mutated (β39; CAG→UAG) human β-globin gene constructs. Genomic DNA was extracted from MEL cells transfected with a β-globin construct as specified above each lane, where each number indicates an independent cell clone. Untransfected MEL (t- MEL) and human genomic DNA were used as negative and positive controls, respectively. DNA was digested with EcoNI plus KpnI enzymes and blots were hybridized with a [α-³²P]dCTP-labeled probe of the human β-globin gene that recognizes a 5.0 kb fragment integrated in the murine genome or a 6.1 kb fragment in the human genomic DNA. MEL cell clones selected for further analysis are indicated in bold. (B) Transgene copy number for each selected MEL cell clone was determined by quantitative PCR using primers specific for human β-globin gene and the endogenous murine Thy1 gene. Quantification was performed by the relative standard curve method. Chart shows the mean ± standard deviation qPCR data from three independent experiments.