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| Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis |
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| Author(s): Donald RGK, Roos DS |
| Source: MOLECULAR AND BIOCHEMICAL PARASITOLOGY Volume: 91 Issue: 2 Pages: 295-305 Published: MAR 15 1998 |
| Times Cited: 54 References: 47 |
| Abstract: The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene of the protozoan parasite Toxoplasma gondii encodes a safe, practical genetic marker suitable for both positive and negative selection. Taking advantage of the ability to control homologous versus nonhomologous recombination in haploid T. gondii tachyzoites by manipulating the length of homologous DNA sequence, we have explored the possibility of 'hit-and-run' mutagenesis to introduce gene knock-outs (or allelic replacements) at loci for which no known selection or screen is available. Using the uracil phosphoribosyl transferase (UPRT) locus as a target, a genomic clone containing approximate to 8 kb encompassing the UPRT gene (but lacking essential coding sequence) was fused to a cDNA-derived HXGPRT 'minigene', which lacks sufficient continguous genomic sequence for homologous recombination. After transfection of circular plasmid DNA, positive selection for HXGPRT activity identified stable transformants, > 30% of which were found to have integrated at the UPRT locus as 'pseudodiploids' (produced by single-site homologous recombination between the circular plasmid and genomic DNA). Upon removal of mycophenolic acid, resolution of pseudodiploids by spontaneous intrachromosomal homologous recombination was selected using 6-thioxanthine, yielding a 1:1 ratio of UPRT knock-out parasites to wild-type revertants, at frequencies of approximate to 10(-6) per parasite doubling. Applications of 'hit-and-run' technology relative to other gene targeting strategies are discussed. (C) 1998 Elsevier Science B.V. All rights reserved. |
| Document Type: Article |
| Language: English |
| Reprint Address: Roos, DS (reprint author), Univ Penn, Dept Biol, Philadelphia, PA 19104 USA |
Addresses:
1. Univ Penn, Dept Biol, Philadelphia, PA 19104 USA |
| Publisher: ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS |
| Subject Category: Biochemistry & Molecular Biology; Parasitology |
| IDS Number: ZG199 |
| ISSN: 0166-6851 |
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