Abstract
Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line mCherry-luc@etramp10.3). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The mCherry-luc@etramp10.3 parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.
Highlights
Transgenic rodent and human malaria parasites expressing fluorescent and bioluminescent proteins are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology and as tools to evaluate anti-parasite inhibitors and vaccines (Othman et al, 2017)
The essential role of ETRAMP10.3 during asexual bloodstage development and its expression in asexual blood-stages and gametocytes is in contrast to expression of the UIS4 protein of the rodent parasites, P. berghei and P. yoelii
The P. berghei and P. yoelii uis4 gene is dispensable for bloodstage development s (Kaiser et al, 2004; Mueller et al, 2005) and there is no evidence that uis4 is expressed in blood-stages either from proteomic or transcriptomic analyses [www.PlasmoDB.org; (Otto et al, 2014)]
Summary
Transgenic rodent and human malaria parasites expressing fluorescent and bioluminescent proteins are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology and as tools to evaluate anti-parasite inhibitors and vaccines (Othman et al, 2017). To create the mCherry-luc@etramp reporter line we used a previously described Cas9 construct (pLf0019), containing the Cas9 expression cassette with a blasticidin (BSD) drugselectable marker cassette (Mogollon et al, 2016) in combination with a sgRNA donor-DNA plasmid (pLf0049).
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