TY - JOUR
T1 - Generating knock-in parasites
T2 - Integration of an ornithine decarboxylase transgene into its chromosomal locus in Leishmania donovani
AU - Roberts, Sigrid C.
AU - Kline, Chelsey
AU - Liu, Wei
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported by Grant R01 AI041622 from the National Institute of Allergy and Infectious Disease .
PY - 2011/6
Y1 - 2011/6
N2 - 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.
AB - 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.
KW - Genetic manipulations
KW - Leishmania
KW - Ornithine decarboxylase
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U2 - 10.1016/j.exppara.2011.02.020
DO - 10.1016/j.exppara.2011.02.020
M3 - Article
C2 - 21354142
AN - SCOPUS:79954422401
SN - 0014-4894
VL - 128
SP - 166
EP - 169
JO - Experimental Parasitology
JF - Experimental Parasitology
IS - 2
ER -