Tetrahymena bifunctional dihydrofolate reductase -...

C - Chemistry – Metallurgy – 12 – N

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C12N 1/10 (2006.01) C12N 9/06 (2006.01) C12N 9/10 (2006.01) C12N 15/52 (2006.01) C12N 15/63 (2006.01) C12N 15/79 (2006.01) C12Q 1/18 (2006.01)

Patent

CA 2623050

A method for producing a ciliate cell with reduced or essentially no dihydrofolate reductase (DHFS) activity or reduced or essentially no thymidylate synthase (TS) activity or both reduced or essentially no dihydrofolate reductase and thymidylate synthase (DHFR-TS) activity is claimed, comprising the steps of a) transforming ciliate cells by inserting a construct containing an allele altering the gene encoding the endogenous DHFR- TS into at least one of the endogenous DHFR-TS genes of the ciliate macronucleus (MAC), b) inducing an allelic assortment process in the transformed ciliate cells to generate cells having the construct inserted in most or all functional DHFR-TS genes of the MAC, and c) identifying the cells generated in step b) by cultivation with or without thymidine.

L'invention concerne un procédé de production d'une cellule ciliée présentant une activité dihydrofolate réductase (DHFS) réduite ou sensiblement inexistante ou une activité thymidylate synthase (TS) réduite ou sensiblement inexistante ou une activité dihydrofolate réductase et thymidylate synthase (DHFR-TS) réduite ou sensiblement inexistante. Le procédé comprend les étapes consistant à : a) transformer des cellules ciliées par insertion d'une construction contenant un allèle modifiant le gène codant pour le DHFR-TS endogène dans au moins un des gènes d'HFR-TS endogènes du macronoyau cilié (MAC), b) induire un procédé d'assortiment allélique dans les cellules ciliées transformées afin de générer des cellules présentant la construction insérée dans la plupart ou dans la totalité des gènes de DHFR-TS fonctionnels du MAC, et c) identifier les cellules générées lors de l'étape b) par culture avec ou de la thymidine ou sans.

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