ISSN:
1550-7408
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
Notes:
In the past few years, genetic manipulation of Plasmodium falciparum has allowed functional studies of many parasite proteins. However, a careful interpretation of gene manipulation results is required, as chromosomal events may occur in vitro independently of the experimental modifications (gene invalidation, overexpression …), and because of the intrinsic mutagenic potential of genetic manipulation, or pleiotropic consequences related to the insertion of exogenous sequences. To ensure that mutant phenotypes arise from the planned mutations, it is necessary to obtain revertants re-expressing the wild-type gene. On this line, we have developed a positive/negative selection strategy allowing in a first step, gene targeting, then excision of the resistance marker. We converted the Toxoplasma gondii DHFR/TS into a tri-functional enzyme following fusion with the Herpes virus type I thymidine kinase (HSV-TK1). Positive selection corresponds to pyrimethamine resistant development, while negative selection acts in presence of the pro-drogue ganciclovir. We used this tool to study RESA, a dense granule protein that associates with spectrin following invasion. Resa gene invalidation was obtained using a targeting plasmid allowing expression of the fusion DHFR/TS/HSV-TK1, and molecular and phenotypic analyses of mutant parasites were performed. Next, wild-type resa gene expression was restored after excision of all the exogenous sequences following phosphorylation of ganciclovir by the HSV thymidine kinase into a toxic metabolite. Phenotypic studies of revertants confirm some functions attributed to RESA protein, and validate the positive/negative strategy in this reverse genetic approach, a useful tool for development of “hit and run” mutagenesis in P. falciparum.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_3_3.x
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