Stable transfection of malaria parasite blood stages

• Published in: Science (American Association for the Advancement of Science) Vol. 268; no. 5215; pp. 1358 - 1362
• Main Authors: Dijk, M.R. van (University of Leiden, Leiden, Netherlands.), Waters, A.P, Janse, C.J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for the Advancement of Science 02-06-1995; American Association for the Advancement of Science; The American Association for the Advancement of Science
• Subjects: Animals; ANTIPROTOZOAIRE; Base Sequence; Biological and medical sciences; Biotechnology; Body Composition; Coding; DNA; DNA Replication; Drug Resistance; Drug Therapy; ELECTROPORACION; ELECTROPORATION; Erythrocytes - parasitology; ETAPAS DEL DESARROLLO ANIMAL; EXPERIMENTACION IN VIVO; EXPERIMENTATION IN VIVO; Fundamental and applied biological sciences. Psychology; Genes, Protozoan; Genetic engineering; Genetic loci; Genetic technics; Genetic transformation; Genetic Vectors; Genetics; Genomics; INFECCION EXPERIMENTAL; INFECCIONES POR PROTOZOOS; INFECTION EXPERIMENTALE; Malaria; Medical research; MEDICAMENT; MEDICAMENTOS; MEDICAMENTOS CONTRA PROTOZOARIOS; Methods. Procedures. Technologies; Molecular Biology; Molecular Sequence Data; Multienzyme Complexes - genetics; Oligonucleotides; Parasites; Plasmids; PLASMODIUM; PLASMODIUM BERGHEI; Plasmodium berghei - drug effects; Plasmodium berghei - genetics; Plasmodium berghei - growth & development; Plasmodium falciparum; Point Mutation; Polymerase chain reaction; PROTOZOOSE; Pyrimethamine - pharmacology; RAT; RATA; Rats; Rats, Wistar; Replication Origin; RESISTANCE AUX PRODUITS CHIMIQUES; RESISTENCIA A PRODUCTOS QUIMICOS; STADE DE DEVELOPPEMENT ANIMAL; Tetrahydrofolate Dehydrogenase - genetics; Thymidylate Synthase - genetics; Transfection; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Plasmid; Protozoa; Plasmodium berghei; Stability; Genetic transformation; Merozoite; Molecular integration; Electroporation; Method; Sporozoa; Vector
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.7761856

Genetic manipulation of malaria parasites would revolutionize the study of this group of pathogens and have implications for vaccine and drug development. This report describes the stable, drug-selectable genetic transformation of the clinically relevant intracellular blood stages of a malaria parasite. A plasmid transfection vector carrying the gene locus that encodes a drug-resistant form of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from the rodent malaria parasite Plasmodium berghei was constructed. Derivatives of this vector were introduced into merozoites of P. berghei by electroporation, and parasites were selected for successful transformation in the rodent host on the basis of resistance to pyrimethamine. The plasmids were present in a circular, unrearranged form that replicated episomally to an observed maximum of 15 copies per cell in drug-resistant populations