Generating knock-in parasites: Integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani

Sigrid C. Roberts, Chelsey Kline, Wei Liu, Buddy Ullman

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Leishmania null mutants created by targeted gene replacement are typically complemented with chimeric episomes harboring the replaced gene in order to validate that the observed phenotype is due to the specific gene deletion. However, the current inventory of available episomes for complementation of genetic lesions in Leishmania is unstable in the absence of drug selection, and levels of gene expression cannot be controlled, especially in vivo. To circumvent this impediment, a strategy to re-introduce the targeted gene into the original chromosomal locus to generate " knock-in" parasites within selectable null backgrounds has been developed. A genomic fragment encompassing the ornithine decarboxylase locus and lacking heterologous DNA sequences was transfected into ornithine decarboxylase-deficient Leishmania donovani. The construct randomly integrated into either chromosomal allele by homologous recombination restoring polyamine prototrophy and revealing that LdODC was functionally expressed in the knock-in clones. This strategy offers a mechanism for complementing a genetic lesion amenable to positive selection in a manner that facilitates stable gene expression from its original locus in the absence of continuous drug pressure.

Original languageEnglish (US)
Pages (from-to)166-169
Number of pages4
JournalExperimental Parasitology
Volume128
Issue number2
DOIs
StatePublished - Jun 2011

Fingerprint

Leishmania donovani
Ornithine Decarboxylase
Leishmania
Transgenes
Parasites
Plasmids
Gene Expression
Gene Order
Homologous Recombination
Gene Deletion
Polyamines
Pharmaceutical Preparations
Genes
Clone Cells
Alleles
Phenotype
Pressure
Equipment and Supplies

Keywords

  • Genetic manipulations
  • Leishmania
  • Ornithine decarboxylase

ASJC Scopus subject areas

  • Parasitology
  • Immunology

Cite this

Generating knock-in parasites : Integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani. / Roberts, Sigrid C.; Kline, Chelsey; Liu, Wei; Ullman, Buddy.

In: Experimental Parasitology, Vol. 128, No. 2, 06.2011, p. 166-169.

Research output: Contribution to journalArticle

Roberts, Sigrid C. ; Kline, Chelsey ; Liu, Wei ; Ullman, Buddy. / Generating knock-in parasites : Integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani. In: Experimental Parasitology. 2011 ; Vol. 128, No. 2. pp. 166-169.
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