| Autor: |
Wang, Sen, Li, Dongfang, Chen, Fangwei, Jiang, Weijun, Luo, Wanxin, Zhu, Guan, Zhao, Junlong, He, Lan |
| Předmět: |
|
| Zdroj: |
Frontiers in Cellular & Infection Microbiology; 4/6/2022, Vol. 12, p1-11, 11p |
| Abstrakt: |
Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of Babesia duncani , a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene elongation factor-1α (ef-1α) and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the ef-1αB promoter and the reporter gene mCherry and achieved stable transfection through homologous recombination to integrate the selection marker hDHFR-eGFP into the parasite genome. The potential of this genetic modification method was tested by knocking out the thioredoxin peroxidase-1 (TPX-1) gene, and under the drug pressure of 5 nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the TPX-1 knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for B. duncani , which may facilitate gene function research and vaccine development of B. duncani. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
|
