Establishment of a transient transfection system for Babesia sp. Xinjiang with the use of homologous promoters

BackgroundBabesia species, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, including Plasmodium falciparum, Trypanosoma cruzi, Cryptosporidium parvum, Theileria annulata, Theileria parva, Babesia bovis, Babesia bigemina, Babesia ovata, Babesia gibsoni, and Babesia ovis. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis mainly in China, the efficiency of these techniques remain unclear. MethodsFirst, a plasmid bearing the elongation factor-1 alpha promoter, as well as the firefly luciferase reporter gene and rap stop region were transfected into Bxj by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program setting were evaluated to confirm the best for Bxj transient transfection and a series of different amounts of plasmid DNA were applied to generate relatively high luminescence values. Finally, the activities of four promoters derived from Bxj were evaluated using the developed transient transfection system. ConclusionsIn this study, a transient transfection system for Bxj was optimized. These findings provided critical information for Bxj genetic manipulation, as an essential tool to identify virulence factors and to further elucidate the basic biology of pathogens.

Transient Transfection of the Zoonotic Parasite Babesia microti

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5′-actin promoter, program FA100, and 50 μg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

The Coding sequence of firefly luciferase reporter gene affects specific hyperexpressionArabidopsis thaliana cpl1mutant

Forward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles ofCPL1(Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type andcpl1.Here we show that theLUCcoding sequence is responsible for the high expression incpl1,using a classicalRD29a-LUC. Deletion of theLUC3’-UTR did not change hyperactivation ofLUCincpl1.However, a codon-modifiedLUC(LUC2) produced similar expression levels both in wild type and incpl1. These results indicate that the coding region ofLUCis responsible for thecpl1-specificLUCoverexpression uncoupled with the expression of the endogenous counterpart.