Development of a novel electroporation method for the oyster parasite Perkinsus marinus.
Sci Rep
; 12(1): 19996, 2022 11 21.
Article
em En
| MEDLINE
| ID: mdl-36411330
ABSTRACT
Gene manipulation techniques are fundamental to molecular biology and are continuously being improved. However, gene transfection methods are not established for many unicellular eukaryotes (protists), thereby hindering molecular biological investigations. The oyster parasite Perkinisus marinus is one of the few protists with established gene transfection and drug selection. Nevertheless, the present protocols are tedious, requiring a specific electroporator and pulse conditions which limits the accessibility of this technique across different research groups. Here, we present alternative buffer and electroporation conditions that make the protocol less restrictive. We revealed the pulse condition that enables the introduction of plasmids into P. marinus cell using Ingenio electroporation buffer and NEPA21 electroporator. We found that number of cells and plasmid concentration were critical parameters for the electroporation system. We also constructed a simpler expression plasmid that is removed needless regions for gene expression in the parasite. Our findings resolved the equipment restriction in electroporation of P. marinus and would be a good reference for electroporation in other protists, in particular other Perkinsozoa parasites and core dinoflagellates.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ostreidae
/
Parasitos
/
Dinoflagellida
/
Apicomplexa
Limite:
Animals
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Japão