Toxoplasma gondii: Stable Complementation of sag1 (p30) Mutants Using SAG1 Transfection and Fluorescence-Activated Cell Sorting

K. Kim, J. C. Boothroyd

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Toxoplasma gondii and the related Apicomplexan protozoan pathogens, Plasmodium, Cryptosporidium, and Eimeria, are obligate intracellular parasites which cause severe disease in their hosts. The recent development of transient transfection of Toxoplasma permits the development of strategies utilizing "reverse genetics" to identify molecules critical to parasite survival within the host. We have utilized transfection of Toxoplasma tachyzoites to stably complement a sag1 (or p30) mutant that does not make detectable SAG1. Transfection of mutants with the wild-type SAG1 gene resulted in transient expression of SAG1 in approximately 15-20% of the transfected population. Stable transformants were enriched by repeated sorting of live parasites using a fluoroscein-labeled monoclonal antibody specific for SAG1. Cloned recombinant parasites expressed SAG1 at wild-type levels and maintained expression for over 5 months after transfection (∼ 300 divisions). Cloned transformants (which proved to be siblings) carried both the mutated gene and one copy of the transfected gene which had inserted randomly into the Toxoplasma genome.

Original languageEnglish (US)
Pages (from-to)46-53
Number of pages8
JournalExperimental Parasitology
Volume80
Issue number1
DOIs
StatePublished - Feb 1995
Externally publishedYes

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Toxoplasma
Transfection
Flow Cytometry
Parasites
Genes
Eimeria
Reverse Genetics
Cryptosporidium
Plasmodium
Monoclonal Antibodies
Genome
Population

ASJC Scopus subject areas

  • Parasitology
  • Immunology
  • Infectious Diseases

Cite this

Toxoplasma gondii : Stable Complementation of sag1 (p30) Mutants Using SAG1 Transfection and Fluorescence-Activated Cell Sorting. / Kim, K.; Boothroyd, J. C.

In: Experimental Parasitology, Vol. 80, No. 1, 02.1995, p. 46-53.

Research output: Contribution to journalArticle

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