STABLE TRANSGENE EXPRESSION IN PLASMODIUM-FALCIPARUM

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodium falciparum were constructed. These consist of a selecta ble gene cassette comprising the Toxoplasma gondii dihydrofolate reduc tase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethami ne resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falc iparum calmodulin promoter sequences and the P. falciparum histidine r ich protein 2 3' region. Also, each vector includes a different expres sion cassette driven by various Plasmodium transcriptional control seq uences. Initially, the chloramphenicol acetyl transferase (CAT) report er gene was cloned into the expression site of two vectors, pCC6-CAT a nd pCC13-CAT, which were identical except for the orientation of the e xpression cassette with respect to the selectable gene cassette. Appro ximately 8-fold more CAT activity was detected when the direction of t ranscription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stab le transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All oth er plasmids were designed with the cassettes in a head to head orienta tion. With the exception of pCC6-CAT and a second vector pHC4-CAT, sta ble transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first tim e vectors for the stable expression in Plasmodium parasites of transge nes other than a selectable marker have been described. (C) 1997 Elsev ier Science B.V.