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

2020 ◽  
Author(s):  
Xiaoxing Wang ◽  
Jinming Wang ◽  
Junlong Liu ◽  
Jifei Yang ◽  
LV Zhaoyong ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Elongation Factor ◽  
Firefly Luciferase ◽  
Transient Transfection ◽  
Babesia Bovis ◽  
Luciferase Reporter ◽  
Babesia Bigemina ◽  
Luciferase Reporter Gene ◽  
Babesia Gibsoni ◽  
Firefly Luciferase Reporter Gene

Abstract 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.

scholarly journals Transient Transfection of the Zoonotic Parasite Babesia microti

Pathogens ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 108 ◽  
Author(s):  
Mingming Liu ◽  
Shengwei Ji ◽  
Mohamed Abdo Rizk ◽  
Paul Franck Adjou Moumouni ◽  
Eloiza May Galon ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Firefly Luciferase ◽  
Transient Transfection ◽  
Babesia Microti ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Transfection Method ◽  
Firefly Luciferase Reporter ◽  
Zoonotic Parasite ◽  
Firefly Luciferase Reporter Gene

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.

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

2017 ◽  
Author(s):  
Hisashi Koiwa ◽  
Akihito Fukudome
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Coding Region ◽  
Luciferase Reporter Gene ◽  
Coding Sequence ◽  
Firefly Luciferase Reporter ◽  
Endogenous Genes ◽  
Firefly Luciferase Reporter Gene

AbstractForward 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.

scholarly journals Identification of Inhibitors of Bacterial Transcription/Translation Machinery Utilizing a Miniaturized 1536-Well Format Screen

2001 ◽  
Vol 6 (4) ◽  
pp. 233-243 ◽  
Author(s):  
Ilona Kariv ◽  
Hong Cao ◽  
Phillip D. Marvil ◽  
Ekaterina V. Bobkova ◽  
Yuri E. Bukhtiyarov ◽  
...  
Keyword(s):  
Multicomponent System ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Data Set ◽  
Vitro Assay ◽  
Bacterial Transcription ◽  
Firefly Luciferase Reporter ◽  
Firefly Luciferase Reporter Gene

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC50 values derived for this assay in a 1536-well format and 384-well format.

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