Stable transfection of the diplomonad parasite Spironucleus salmonicida

• Eukaryotic microbes are highly diverse and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has mainly been studied due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad casuing systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3xHA, 2xOLLAS, 3xMYC) and purification of proteins (2xStrepII-FLAG or SBP-GST) under the control of native or constitutive promoters. Three selectable markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr) or neomycin phosphotransferase (nptII) were successfully applied for generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope-tagged six proteins and confirmed their expression by Western Blot. Next, we demonstrated the utility of these vectors by recording the sub-cellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we describe the creation of a S. salmonicida double transfectant suitable for co-localization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific as well as general diplomonad traits, benefiting research on both Giardia and Spironucleus.

Ämnesord

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Cellbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Cell Biology (hsv//eng)

Nyckelord

Biology with specialization in Microbiology

Biologi med inriktning mot mikrobiologi

Biology with specialization in Molecular Cell Biology

Biologi med inriktning mot molekylär cellbiologi

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)