ArticleProceedings of the National Academy of Sciences of the United States of America1991
Double targeted gene replacement for creating null mutants.
Article in Proceedings of the National Academy of Sciences of the United States of America, 1991. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 119 papers.
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119 citing papers in PubMed, 322 citations in OpenAlex.
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- Combined gene deletion of dihydrofolate reductase-thymidylate synthase and pteridine reductase in Leishmania infantum.PLoS neglected tropical diseases · 2021Article
- Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival.Nature communications · 2021Article
- Deletion of Glutamine Synthetase Gene Disrupts the Survivability and Infectivity ofFrontiers in cellular and infection microbiology · 2021Article
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- A cytoskeletal protein complex is essential for division of intracellular amastigotes ofThe Journal of biological chemistry · 2020Article
- Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model.Parasites & vectors · 2020Article
- Essential gene prediction using limited gene essentiality information-An integrative semi-supervised machine learning strategy.PloS one · 2020Article
- Review
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- Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania.Methods in molecular biology (Clifton, N.J.) · 2020Article
- The Novel Serine/Threonine Protein Kinase LmjF.22.0810 fromBiomolecules · 2019Article
- Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage inmSphere · 2019Article
59 more citing papers are in PubMed but not listed here.
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.
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