TY - JOUR
T1 - Toxoplasma gondii
T2 - Stable Complementation of sag1 (p30) Mutants Using SAG1 Transfection and Fluorescence-Activated Cell Sorting
AU - Kim, Kami
AU - Boothroyd, John C.
PY - 1995/2
Y1 - 1995/2
N2 - 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.
AB - 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.
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U2 - 10.1006/expr.1995.1006
DO - 10.1006/expr.1995.1006
M3 - Article
C2 - 7821410
AN - SCOPUS:0028848059
SN - 0014-4894
VL - 80
SP - 46
EP - 53
JO - Experimental Parasitology
JF - Experimental Parasitology
IS - 1
M1 - 71006
ER -