Finally, cells were mounted with ProLong AntiFade Kit from Molecular Probes (Invitrogen). The stained cells were examined using a Nikon selleck chemicals llc Eclipse E600 microscope and a Nikon Digital camera DXM1200. Results The PTC introduced by the frameshift mutation 3905insT is insensitive to NMD and NAS Four patients (P1�CP4) carrying the 3905insT (c. 3773_3774insT) mutation on one allele and the F508del (p.Phe508del) mutation on the other allele were selected to produce EBV-transformed lymphoblastoid cell cultures. Primers were designed to amplify a region containing the codon for F508del, yielding either a 120-bp fragment for the 3905insT allele or a 117-bp fragment for the F508del allele (Figure 1a). As even slight differences in amplification efficiency between the two fragments can considerably influence quantification results, standard curves were created by the LightCycler software for both cDNAs.

The obtained curves gave very similar slopes for both cDNAs, indicating that the two fragments were amplified with nearly the same efficiency (Figure 1b). Extracted RNA from cell cultures was then used to perform real-time RT�CPCR on the LightCycler using HEX-labeled primers. Amplification was stopped in the exponential phase of the reaction and the amplification products were subsequently separated by capillary electrophoresis on an ABI3100 genetic analyzer. The GeneMapper software 3.5 was then used to integrate the area under the peaks corresponding to the 3905insT (120bp) and the F508del cDNA (117bp). As the F508del mutation is not expected to be affected by NMD, 3905insT transcript levels can be quantified relative to F508del levels.

With the exception of P4 (26%), we obtained relative 3905insT mRNA proportions around 50% with values ranging between 45% (P3) and 54% (P1), indicating that the 3905insT allele produces nearly equal transcript levels compared with the F508del allele in P1, P2, and P3 (Figure 1c). These results strongly suggest that the PTC introduced by the frameshift mutation 3905insT does not cause instability of the corresponding mRNA through NMD. Figure 1 (a) Schematic representation of the RT�CPCR reaction that was performed to discriminate between the F508del and the 3905insT mRNA. Primers were designed to amplify the region containing the codon for phenylalanine at position 508 of the CFTR protein. …

To verify whether the 3905insT mutation is capable of triggering, an NAS response by inducing skipping of the PTC-containing exon, a region of 817bp encompassing exons 19�C24 was amplified by RT�CPCR. Brefeldin_A Skipping of exon 20 would be expected to result in a 156-bp shorter fragment of 661bp. However, neither the RNA from EBV lymphocytes (Figure 2a) nor the RNA from nasal epithelial cells carrying the 3905insT on one allele (Figure 2b) showed an indication for exon 20 skipping.