Characterization of Translational Repression in P. yoelii Sporozoites

Wild-type and transgenic P. yoelii sporozoites (PY17X_1354300::GFP, PyUIS12::GFP) were subjected to live fluorescence assays and/or an indirect immunofluorescence assay (IFA)^{72 (link)} to characterize the extent of translational repression of these candidates. For live fluorescence microscopy of PyUIS12::GFP, freshly produced salivary gland sporozoites placed on glass slides in VectaShield, overlaid with a cover glass slip, and visualized by fluorescent microscopy using a Zeiss Axioscope A1 with 8-bit AxioCam ICc1 camera and Zen imagine software from the manufacturer. Alternatively, fresh salivary gland sporozoites were fixed in 10% v/v formalin for 10 min, and then air dried to a well on a glass slide defined by a hydrophobic coating. Sporozoites were treated for IFA using either rabbit polyclonal anti-PyUIS4 (antigen consisting of AA80-224, diluted 1:1000), produced by Pocono Rabbit Farm and Laboratory, Canadensis, PA) or rabbit polyclonal anti-GFPmut2 (diluted 1:1000) as primary antibodies and anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 as a secondary antibody (diluted 1:500).

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Lindner S.E., Swearingen K.E., Shears M.J., Walker M.P., Vrana E.N., Hart K.J., Minns A.M., Sinnis P., Moritz R.L, & Kappe S.H. (2019). Transcriptomics and proteomics reveal two waves of translational repression during the maturation of malaria parasite sporozoites. Nature Communications, 10, 4964.

Publication 2019

Axioscope A1 AxioCam ICc1 camera

Alexa fluor 488 Anti igg Antibodies Antibody Antigen Assays Fluorescence Fluorescence microscopy Formalin Indirect immunofluorescence assay Microscopy Rabbit Repression Salivary gland Sporozoites Transgenic Translational

Corresponding Organization : Pennsylvania State University

Other organizations : Institute for Systems Biology, Johns Hopkins University

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Variable analysis

independent variables

Genetic manipulation of P. yoelii parasites (wild-type vs. transgenic PyUIS12::GFP and PY17X_1354300::GFP)

dependent variables

Extent of translational repression of PyUIS12 and PY17X_1354300 candidates, as characterized by live fluorescence assays and/or indirect immunofluorescence assay (IFA)

control variables

Freshly produced salivary gland sporozoites
Sporozoite fixation in 10% v/v formalin for 10 min
Sporozoite air-drying on a well-defined glass slide
Use of primary antibodies (rabbit polyclonal anti-PyUIS4 and anti-GFPmut2)
Use of secondary antibody (anti-rabbit IgG conjugated to Alexa Fluor 488)

controls

Positive control: Wild-type P. yoelii sporozoites
Negative control: Not explicitly mentioned

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