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- Title
Generation of Leishmania Hybrids by Whole Genomic DNA Transformation.
- Authors
Coelho, Adriano C.; Leprohon, Philippe; Ouellette, Marc
- Abstract
Genetic exchange is a powerful tool to study gene function in microorganisms. Here, we tested the feasibility of generating Leishmania hybrids by electroporating genomic DNA of donor cells into recipient Leishmania parasites. The donor DNA was marked with a drug resistance marker facilitating the selection of DNA transfer into the recipient cells. The transferred DNA was integrated exclusively at homologous locus and was as large as 45 kb. The independent generation of L. infantum hybrids with L. major sequences was possible for several chromosomal regions. Interfering with the mismatch repair machinery by inactivating the MSH2 gene enabled an increased efficiency of recombination between divergent sequences, hence favouring the selection of hybrids between species. Hybrids were shown to acquire the phenotype derived from the donor cells, as demonstrated for the transfer of drug resistance genes from L. major into L. infantum. The described method is a first step allowing the generation of in vitro hybrids for testing gene functions in a natural genomic context in the parasite Leishmania. Author Summary: Leishmania spp. are pathogenic protozoa characterized by a substantial diversity in pathogenesis and virulence despite their considerable synteny at the genome level. The existence of genetic exchange was recently proven experimentally in the sand fly vector where hybrid parasites were isolated and generated. Here, we show the feasibility of generating Leishmania hybrids by electroporating genomic DNA of donor cells into recipient Leishmania parasites. This methodology was made possible by introducing a drug resistance marker in the donor Leishmania cells that could be used for selecting recombinant recipient parasites. Integrations of exogenous DNA fragments as large as 45 kb were possible for several chromosomal regions and took place at homologous loci in recipient Leishmania strains. Our observations are the first step for the generation of in vitro hybrids for assessing gene function under natural genomic contexts and this technology may be applicable to other pathogens.
- Subjects
LEISHMANIA; DNA; GENE targeting; PATHOGENIC protozoa; SAND flies; MICROBIAL genes
- Publication
PLoS Neglected Tropical Diseases, 2012, Vol 6, Issue 9, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0001817