Gene insertion by direct ligation in vitro

Details Gene insertion by direct ligation in vitro Gene insertion by direct ligation in vitro

C - Chemistry, Metallurgy

12

N

C12N 15/85 (2006.01) C07K 14/435 (2006.01) C12N 7/00 (2006.01) C12N 15/86 (2006.01) C12N 15/866 (2006.01)

Patent

CA 2141206

A method is described for construction recombinant double stranded DNA viruses, especially baculoviruses and granulosis viruses, by the direct ligation of DNA fragments in vitro. Also described are direct ligation virus vectors, which are insect viruses that have been modified by the insertion of at least one recognition site for a specific restriction endonuclease which does not cut the viral genome, so as to facilitate the insertion of foreign DNA segments by DNA ligation in vitro, and to the recombinant viruses formed by this direct ligation. Further described are modular expression vectors (plasmids) that are designed to facilitate the assembly of gene expression cassettes or other DNA fragments into virus insertion modules (as shown in the figure) which are inserted subsequently into direct ligation virus vectors at predefined sites and in a predifined orientation by ligation in vitro, and to recombinant viruses derived from said modular expression vectors.

L'invention concerne un procédé de construction de virus d'ADN de recombinaison bicaténaire, en particulier des baculovirus et des virus granulosis, par liaison directe de fragments d'ADN in vitro. L'invention porte également sur des vecteurs viraux de liaison directe qui sont des virus d'insecte modifiés par insertion d'au moins un site de reconnaissance d'une endonucléase de restriction spécifique ne coupant pas le génome viral, de manière à faciliter l'insertion de segments d'ADN étrangers par liaison de l'ADN in vitro, ainsi que sur les virus de recombinaison formés par cette liaison directe. Des vecteurs d'expression modulaires (plasmides) conçus pour faciliter l'assemblage des cassettes d'expression de gènes ou d'autres fragments d'ADN dans des modules d'insertion de virus qui sont insérés ultérieurement dans des vecteurs viraux de liaison directe sur des sites prédéfinis, et selon une orientation prédéfinie par liaison in vitro, ainsi que des virus de recombinaison dérivés desdits vecteurs d'expression modulaires sont également décrits.

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