||Contract AIR3-CT94-2487 - European Commission
||Delsert Claude, Cancela Da Fonseca Leonor
||Pathology, Mussel-specific expression vectors, Cell culture, Mytilus galloprovincialis, Gene, Genetic marker
||The main objective of this project was to develop the tools for the in-depth understanding of mollusc biology and pathology. Such a goal has been reached in other biological systems through the development of molecular biology and cell culture. Indeed, development of a mollusc cell culture system is an essential step to approach many of the problems related to mollusc pathology, in particular to use molecular biology tools to study viral infection of these organisms. No continous cell line is available from bivalves and while cell cultures have been obtained from these organisms they are difficult to develop and labor intensive, survival cultures being maintained only for a few weeks. Our goal has been to study in detail many of the parameters involved in maintaining primary cell cultures from bivalve molluscs in an effort to ease the burden associated with maintenance of these ce Ils in culture. We then studied different ways to transform primary cell culture, in an effort to obtain continuous cell lines. The initial part of this project was devoted to the development of mussel-specific expression vectors in order to help the transformation of mollusc primary cell culture. This approach required (i) the development of mollusc-specific expression vectors and (ii) the establishment of optimal conditions for primary cell cultures and (3i) transfection of expression vectors and detection of the expression of reporter genes and (4i) expression of oncogenes in primary cell cultures. A mussel genomic DNA library was built and was screened using the actin A3 gene of Bombyx mori as a probe. Clones corresponding to the mussel actin gene family were isolatep and characterized. The promoter regions of five of these genes were subcloned and used ta build mussel-specific expression vectors carrying the B-galactosidase or Luciferase reporter sequence. In addition, the large T antigen of the simian SV40 virus, a potent oncogene in mammalian systems, was inserted in one of the expression vectors. Expression vectors were then transfected in primary cell cultures which were maintained in optimal culture conditions, as previously defined. Some success has been recorded as shown by detection of the reporter gene activity although the rate of transfection remained somehow low. Meanwhile, further experiments were performed (i) to determine transcription parameters such as the transcription start site and the tissue-specificity for each gene (ii) to analyze the level of expression of these genes at various developmental stages (iii) to look for cellular transformation by using carcinogenic drugs to treat different primary cell cultures or mollusc larvaes maintained in cell culture media (4i) to assess transient expression of a reporter gene by electroporation of axenic oyster larvae (5i) to uncover through a cDNA subtraction approach, a number of genes differentially expressed du ring in vitro cell culture as compared to gene expression in non-dispersed tissues. Data concerning the molecular biology of molluscs and our observations regarding the obtention and transfection of mollusc primary cell cultures are discussed.