Modifications génétiques animales et végétales : méthodes de transgénèse et expression des transgènes.

Abstract

La transgénèse est un moyen essentiel pour étudier le rôle des gènes dans l’expression des fonctions biologiques ainsi que leur fonctionnement. Elle permet également d’envisager des applications biotechnologiques diverses. Bien que vieille de vingt ans chez les animaux et de dix-sept ans chez les végétaux, elle souffre encore de limites techniques qui sont progressivement repoussées. L’expression des transgènes est souvent mal contrôlée. C’est surtout le cas lorsque des gènes étrangers sont ajoutés dans des sites quelconques des génomes. L’expression est considérée comme satisfaisante lorsque le transgène est exprimé dans toutes les lignées, d’une manière qui respecte la spécificité du promoteur utilisé et, dans l’idéal, en fonction du nombre de copies intégrées. De nouveaux outils, qu’il est encore nécessaire de perfectionner, permettent de mieux utiliser la transgénèse pour des études fondamentales et de développer diverses applications dans les domaines médical et agronomique.

Gene transfer to generate transgenic animals and plants has been performed for the first time in 1980-1982 and 1983 respectively. Transgenesis has been extended to other mammals (rat, rabbit, pig, goat, sheep and cow), to lower vertebrates (chicken, fish, xenopus) and to invertebrates (Drosophila, C. elegans). Direct gene microinjection into one-cell embryos, originally, defined in mouse, is the most frequently used method for most animal species. Gene transfer into sperms or oocytes is only marginal. The use of the animal cloning by transfer of nuclei from previously transfected cells led to the generation of transgenic sheep and goats. Gene replacement by homologous recombination has been restricted essentially to mouse until 1999. It is based on the use of embryonic stem cells (ES cells) and the generation of chimeric animals. The technique of embryo cloning allowed in 1999 the generation of sheep having a gene specifically replaced by homologous recombination. Transgenesis in plants appeared soon as relatively easy to manage, at least in some species, via the Ti vector from Agrobacterium tumefaciens and later by using biolistic. The expression of transgenes is often badly controlled. This is mainly the case when foreign genes are added at any site in genomes. A satisfactory expression is observed when the transgene is expressed in all the lines, according to the specificity of the promoter used and ideally as a function of copy number. These conditions are often met when the foreign gene is flanked by long genomic sequences or when it is inserted into a selected site. Vectors allow a conditional expression of transgenes which depends on exogenous inducers, such as tetracycline, having no action on the host genes. Several systems allow a transgene to interrupt specifically the expression of a cellular gene at its mRNA level or via the overexpression of a transdominant negative protein. These tools which still have to be improved favour the use of transgenesis for basic studies and for various biotechnological applications in medecine and agriculture.