One-step generation of double-allele gene replacement mutants in Leishmania donovani

International Journal for Parasitology

Volumn 39, Issue 5, 2009, Pages 541-546

  Ommen, Gabi ^a   Lorenz, Sabine ^a   Clos, Joachim ^a  

^a BERNHARD NOCHT INSTITUTE FOR TROPICAL MEDICINE   (Germany)

Author keywords

Co transfection; Gene replacement; Homologous recombination; Leishmania donovani

Indexed keywords

ALLELE; ANTIBIOTIC RESISTANCE; GENE EXPRESSION; LEISHMANIASIS; MUTATION; PROTOZOAN; RECOMBINATION; REPLACEMENT;

ALLELES; ANIMALS; BLOTTING, WESTERN; CELLS, CULTURED; DNA, PROTOZOAN; DNA, RECOMBINANT; DRUG RESISTANCE, MICROBIAL; FEASIBILITY STUDIES; GENE TARGETING; GENES, PROTOZOAN; LEISHMANIA DONOVANI; MACROPHAGES; MUTAGENESIS, INSERTIONAL; PROTOZOAN PROTEINS; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; TRANSFECTION;

PROTOZOAL DNA; PROTOZOAL PROTEIN; RECOMBINANT DNA;

ALLELE; ANIMAL; ANTIBIOTIC RESISTANCE; CELL CULTURE; FEASIBILITY STUDY; GENE; GENE EXPRESSION REGULATION; GENE TARGETING; GENETIC TRANSFECTION; GENETICS; GROWTH, DEVELOPMENT AND AGING; LEISHMANIA DONOVANI; MACROPHAGE; PARASITOLOGY; PROCEDURES; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; WESTERN BLOTTING;

EID: 59749090972     PISSN: 00207519     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.ijpara.2008.10.009     Document Type: Article

References (25)

1. Beverley S.M. Protozomics: trypanosomatid parasite genetics comes of age. Nat. Rev. Genet. 4 (2003) 11-19
2. Brandau S., Dresel A., and Clos J. High constitutive levels of heat-shock proteins in human-pathogenic parasites of the genus Leishmania. Biochem. J. 310 (1995) 225-232
3. Clos J., and Brandau S. pJC20 and pJC40-two high-copy-number vectors for T7 RNA polymerase-dependent expression of recombinant genes in Escherichia coli. Protein Expr. Purif. 5 (1994) 133-137
4. Cruz A., and Beverley S.M. Gene replacement in parasitic protozoa. Nature 348 (1990) 171-173
5. Cruz A., Coburn C.M., and Beverley S.M. Double targeted gene replacement for creating null mutants. Proc. Natl. Acad. Sci. USA 88 (1991) 7170-7174
6. Genest P.A., ter Riet B., Dumas C., Papadopoulou B., van Luenen H.G., and Borst P. Formation of linear inverted repeat amplicons following targeting of an essential gene in Leishmania. Nucleic Acids Res. 33 (2005) 1699-1709
7. Gueiros-Filho F.J., and Beverley S.M. Selection against the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major. Mol. Cell. Biol. 16 (1996) 5655-5663
8. Hong K., Ma D., Beverley S.M., and Turco S.J. The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits. Biochemistry 39 (2000) 2013-2022
9. Hoyer C., Zander D., Fleischer S., Schilhabel M., Kroener M., Platzer M., and Clos J. A Leishmania donovani gene that confers accelerated recovery from stationary phase growth arrest. Int. J. Parasitol. 34 (2004) 803-811
10. Hubel A., Krobitsch S., Horauf A., and Clos J. Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite. Mol. Cell. Biol. 17 (1997) 5987-5995
11. Kapler G.M., Coburn C.M., and Beverley S.M. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol. Cell. Biol. 10 (1990) 1084-1094
12. Krobitsch S., Brandau S., Hoyer C., Schmetz C., Hübel A., and Clos J. Leishmania donovani heat shock protein 100: characterization and function in amastigote stage differentiation. J. Biol. Chem. 273 (1998) 6488-6494
13. Krobitsch S., and Clos J. Cross-species homologous recombination in Leishmania donovani reveals the sites of integration. Mol. Biochem. Parasitol. 107 (2000) 123-128
14. Laban A., and Wirth D.F. Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene. Proc. Natl. Acad. Sci. USA 86 (1989) 9119-9123
15. Legare D., Richard D., Mukhopadhyay R., Stierhof Y.D., Rosen B.P., Haimeur A., Papadopoulou B., and Ouellette M. The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase. J. Biol. Chem. 276 (2001) 26301-26307
16. Nascimento M., Zhang W.W., Ghosh A., Houston D.R., Berghuis A.M., Olivier M., and Matlashewski G. Identification and characterization of a protein-tyrosine phosphatase in Leishmania: involvement in virulence. J. Biol. Chem. 281 (2006) 36257-36268
17. Nolan T.J., and Herman R. Effects of long-term in vitro cultivation on Leishmania donovani promastigotes. J. Protozool. 32 (1985) 70-75
18. Papadopoulou B., and Dumas C. Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania. Nucleic Acids Res. 25 (1997) 4278-4286
19. Robinson K.A., and Beverley S.M. Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania. Mol. Biochem. Parasitol. 128 (2003) 217-228
20. Sambrook J., Fritsch E.F., and Maniatis T. Molecular Cloning (1989), Cold Spring Harbor Laboratory Press, Plainview, NY
21. Schlueter A., Wiesgigl M., Hoyer C., Fleischer S., Klaholz L., Schmetz C., and Clos J. Expression and subcellular localization of Cpn60 protein family members in Leishmania donovani. Biochim. Biophys. Acta 1491 (2000) 65-74
22. Scholz, A., 2008. Doctoral thesis, University of Hamburg, Hamburg. Available from: .
23. Tobin J.F., Laban A., and Wirth D.F. Homologous recombination in Leishmania enriettii. Proc. Natl. Acad. Sci. USA 88 (1991) 864-868
24. Ullu E., Tschudi C., and Chakraborty T. RNA interference in protozoan parasites. Cell. Microbiol. 6 (2004) 509-519
25. Wiesgigl M., and Clos J. Heat shock protein 90 homeostasis controls stage differentiation in Leishmania donovani. Mol. Biol. Cell 12 (2001) 3307-3316