Stable transfection system for Babesia sp. Xinjiang

Background Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers. Methods We evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites. Results Transfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months. Conclusions In our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj. Graphical abstract

miR-34a mimic or pre-mir-34a, which is the better option for cancer therapy? KatoIII as a model to study miRNA action in human gastric cancer cells

Background Aberrantly expressed microRNAs play important roles in gastric tumorigenesis. However, use of miRNAs as a therapeutic option in gastric cancer still remains as a challenging problem. Methods We performed transient transfection of miR-34a-5p mimic and stable transfection of pre-mir-34a into KatoIII cells. Then, we evaluated the effect of transfected miRNAs on numerous cellular and molecular processes. Results Following transient transfection of miR-34a-5p mimic at 25 nM—a commonly used concentration—into KatoIII cells, inhibition of two target genes expression, namely Notch1 and β-catenin, was not observed, but a non-significant marginal increase of these genes was detected. No changes were detected in the percentage of apoptotic cells as well as in CD44 + and EpCAM + cells after 25 nM miR-34a-5p mimic transfection. Interestingly, stable transfection of pre-mir-34a into KatoIII cells (named as KatoIII-pGFPC1-34a cells) caused a significant repression in β-catenin protein and Notch1 mRNA levels (p < 0.05 and p < 0.01, respectively) relative to equivalent control (KatoIII- pGFPC1-empty cells). The percentage of CD44 + cells in the KatoIII-pGFPC1-34a cells (< 40%) was significantly lower than that in control cells (~ 95%) (p < 0.05). An increase of ~ 3.5% in apoptotic cells and a slower proliferation rate were detected in KatoIII-pGFPC1-34a cells. Conclusions Our study revealed that the effect of miR mimic in target gene repression can be dependent to its concentration as well as to the cell type. Meanwhile, our findings further support a regulatory function for pre-miRNAs in target repression and will help to develop effective therapeutic strategies in cancer treatment.

Stable transfection in the protist Corallochytrium limacisporum allows identification of novel cellular features among unicellular relatives of animals

ABSTRACTThe evolutionary path from protists to multicellular animals remains a mystery. Recent work on the genomes of several unicellular relatives of animals has shaped our understanding of the genetic changes that may have occurred in this transition. However, the specific cellular modifications that took place to accommodate these changes remain unclear. Functional approaches are now needed to unravel how different cell biological features evolved. Recent work has already established genetic tools in three of the four unicellular lineages closely related to animals (choanoflagellates, filastereans, and ichthyosporeans). However, there are no genetic tools available for Corallochytrea, the lineage that seems to have the widest mix of fungal and metazoan features, as well as a complex life cycle. Here, we describe the development of stable transfection in the corallochytrean Corallochytrium limacisporum. Using a battery of cassettes to tag key cellular components, such as nucleus, plasma membrane, cytoplasm and actin filaments, we employ live imaging to discern previously unknown biological features of C. limacisporum. In particular, we identify two different paths for cell division—binary fission and coenocytic growth—that reveal a non-linear life cycle in C. limacisporum. Additionally, we found that C. limacisporum is binucleate for most of its life cycle, and that, contrary to what happens in most eukaryotes, nuclear division is decoupled from cell division. The establishment of these tools in C. limacisporum fills an important gap in the unicellular relatives of animals, opening up new avenues of research with broad taxon sampling to elucidate the specific cellular changes that occurred in the evolution of animals.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.