The promoter of the human cystic fibrosis transmembrane conductance regulator gene directing SV40 T antigen expression induces malignant proliferation of ependymal cells in transgenic mice.

Transgenic mice bearing a human cystic fibrosis transmembrane conductance regulator (CFTR) promoter-SV40 T antigen fusion transgene were generated in order to localize in vivo the potential oncogenesis linked to the tissue-specific activity of the promoter for the CFTR gene. Surprisingly, the only site of tumors resulting from expression of the reporter onc gene was ependymal cells lining the brain ventricles. SV40 T antigen expression in these cells led to a consistent pathology in the first weeks of age: ependymoma and consequent hydrocephaly. Tumor-derived cell lines were established, characterized and shown to originate from SV40 T antigen-induced ependymoma. No pathological alterations were found in other organs, such as lungs and pancreas, in which cystic fibrosis is pathologically manifest in humans. Such transgenic mice and derived cell lines may represent valid models for analysing (1) the role of SV40 T antigen in ependymoma formation and (2) CFTR function in ependymal cells.

In vitro and in vivo effects of glucocorticoids on gene transfer to skeletal muscle.

As a pharmacological approach to potentially improve gene transfer efficiency into skeletal muscle cells, glucocorticoids were shown here to allow efficient transfection of cultured and mouse human myoblasts, human pulmonary A549 cells, but not dog myoblasts, independently of the transfection protocol, the reporter gene and the transcription promoter employed. Transduction with adenovirus was also increased by dexamethasone. Pretreatment of cells 48 h prior to transfection was most effective and was shown to be concentration-dependent. This effect is mediated by binding to the glucocorticoid receptor, but not by glucocorticoid responsive elements present in the vectors. The acute dexamethasone effect could be due to increased plasmid entry into the cells as suggested by Southern blot, whereas the sustained increase of luciferase activity in dexamethasone-treated cultures may be related to intracellular mechanisms following cell entry. In mice in vivo, a similar increase of luciferase activity upon glucocorticoid treatment was found.

Gene transfer into canine myoblasts.

We have developed and characterized cultures of healthy and dystrophic canine myoblasts for the evaluation of various gene transfer protocols. The number of desmin-positive myoblasts was elevated (>>80%) in cultures of myoblasts obtained from different muscle territories, the diaphragm muscle giving rise to the purest cultures. Myoblasts from dogs turned out to be a very convenient source of well transfectable and transducible cells. Transfection with plasmid DNA allowed efficient transgene expression (50% of beta-galactosidase positive cells and about 375 ng luciferase/mg protein after transfection with a calcium phosphate-precipitated plasmid). Infection with high concentrations of adenoviral and retroviral vectors allowed transgene (beta-galactosidase or mini-dystrophin) detection in about 75 to 90% of the canine cells. Therefore, primary dog myoblast cultures represent a useful in vitro model for viral and non-viral gene delivery, as well as for functional evaluation and cell grafting with applications in genetic diseases, vaccination or production of circulating therapeutic proteins.