Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria.

• DOI: 10.1073/pnas.90.24.11703
• Publication Date: 2006-05-31T12:36:45Z
• Authors: R G Donald; D S Roos
• Keywords: Selectable marker; Dihydrofolate reductase; Coding region

To facilitate genetic analysis of the protozoan parasite Toxoplasma gondii, sequences derived from parasite's fused dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene have been used to produce vectors suitable for stable molecular transformation. Mutations introduced into DHFR coding region by analogy with pyrimethamine-resistant malaria confer drug resistance Toxoplasma, providing useful information on structure DHFR-TS enzymes and a powerful selectable marker studies. Depending particular drug-resistance allele employed conditions selection, can be generated either single copy nonhomologous insertion chromosomal DNA or massively amplified transgenes. Frequencies integration are independent transgenes without continued selection. Cointegration reporter adjacent (under control an promoter) shows no loss cointegrated over many generations. By bringing full power bear these studies should greatly development model system Apicomplexan parasites.