Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.

The authors' surnames were incorrectly captured. The correct surnames are Kakavas V.K., Plageras P., Vlachos T.A., Papaioannou A., Noulas V.A.The online version of the original article can be found at

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In the present study we investigated whether single-strand conformational polymorphism (SSCP) and polyacrylamide gel electrophoresis (PAGE) could be used for the identification of the CFTR DeltaF508 gene mutation, which is commonest in the Greek population. Using DNA from patients carrying this mutation, the appropriate 98 bp region of the CFTR gene was amplified by PCR and the reaction products were analysed by non-radioactive SSCP-electrophoresis using silver staining for band visualization and non-denaturating PAGE to confirm the results. SSCP electrophoretic analysis has been optimized for several parameters in order to achieve the best resolution. Single-strand DNA fragments gave a reproducible pattern of bands, characteristic for the particular mutation. Comparison of the obtain patterns with control samples allowed the detection of the DeltaF508 mutation in the patients studied by SSCP assay and these results were confirmed by the independent method of PAGE. Although SSCP and PAGE can be used for detection of this mutation, PAGE resulted in more distinct patterns than SSCP. It is, therefore, proposed that PAGE can be reliably used for the detection and identification of such a mutation in patients provided that suitable controls are available. The applicability of PAGE to identification of the mutation in carriers, particularly useful for population screening, is also discussed.

In the present study we investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) the four most common b-globin gene mutations in the Greek population: IVS-I-110, Cd39, IVS-I-1, and IVS-I-6. Using DNA from 50 b-thalassemic patients and carriers, we amplified by PCR the appropriate 238-bp region of the human bglobin gene, analyzed the reaction products by nondenaturing polyacrylamide gel electrophoresis, and visualized the bands by silver staining. Single-stranded DNA (ssDNA) fragments showed a reproducible pattern of bands that was characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four most common b-globin gene mutations, we were able to predict the mutations present in a quarter of the patients studied. Our predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. We conclude that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in patients, provided that suitable controls are available. Moreover, the method is easy to apply to the identification of mutations in carriers, which makes it particularly useful for population screening.