Overexpression of PfCARL Protein

The full-length coding sequence for pfcarl (PF3D7_1113300) was amplified from genomic DNA using primers pfcarl LocF/pfcarl LocR and subcloned into a pDC2-based expression vector downstream of the P. falciparum calmodulin promoter, yielding the expression plasmid pDC2-PfCAM-pfcarl-FLAG-attP-BSD. This expression plasmid was transfected into the NF54-attB parasite line together with the pINT plasmid for use in integrase-mediated recombination performed with attP × attB (44 (link)). The parasites selected with 2.5 nM WR99210–2 µg/ml blasticidin–250 µg/ml G418 (45 (link)). Parasites were imaged using a confocal UltraView Vox spinning disk (PerkinElmer) and the indicated antibodies.

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LaMonte G., Lim M.Y., Wree M., Reimer C., Nachon M., Corey V., Gedeck P., Plouffe D., Du A., Figueroa N., Yeung B., Bifani P, & Winzeler E.A. (2016). Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance. mBio, 7(4), e00696-16.

Publication 2016

UltraView Vox spinning disk

Antibodies Calmodulin Coding sequence G418 Genomic Integrase Parasites Plasmid Primers Recombination Vector

Corresponding Organization :

Other organizations : University of California, San Diego, Novartis (Singapore), National University of Singapore, Genomics Institute of the Novartis Research Foundation

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Variable analysis

independent variables

Transfection of the expression plasmid pDC2-PfCAM-pfcarl-FLAG-attP-BSD into the NF54-attB parasite line
Integration of the expression plasmid using integrase-mediated recombination performed with attP × attB

dependent variables

Expression of the pfcarl gene
Localization of the PfCARL protein
Imaging of the parasites using a confocal UltraView Vox spinning disk

control variables

Genomic DNA used as template for amplification of the pfcarl coding sequence
PDC2-based expression vector used for cloning the pfcarl gene
P. falciparum calmodulin promoter used to drive the expression of pfcarl
FLAG tag added to the pfcarl gene for detection purposes
AttP-BSD cassette for selection of transfected parasites
NF54-attB parasite line used as the host for transfection
PINT plasmid used for integrase-mediated recombination
Selection of transfected parasites using 2.5 nM WR99210, 2 µg/ml blasticidin, and 250 µg/ml G418

positive control

Not mentioned

negative control

Not mentioned

Annotations

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