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.

The distal location of the iron responsive region of the hepcidin promoter

The response of hepcidin transcription to iron has been repeatedly documented in living mice, but it is difficult to demonstrate the response in ex vivo systems. We have hydrodynamically transfected mice with plasmid constructs composed of a murine hepcidin 1 promoter and fragments of the promoter fused to a firefly luciferase reporter. This method enabled us to quantitate the response of the hepcidin promoter to short-term feeding of a high-iron diet to mice that have been maintained on an iron-deficient diet. We show that the region of the promoter between 1.6 Kb and 1.8 Kb upstream from the start of translation is essential for the response to iron. The promoter region between −260 bp and −1.6 Kb is not essential for the iron responsiveness of hepcidin promoter. The iron-responsive region that we have mapped is the same region required for the in vitro response of HepG2 cells to stimulation with bone morphogenetic proteins and differs from the LPS/IL-6 responsive area.