Transfection of the protozoan parasite Perkinsus marinus.
Ongoing efforts for sequencing the genome of the protozoan parasite Perkinsus marinus, together with functional genomic initiatives, have continued to provide invaluable information about genes and metabolic pathways that not only will increase our understanding of its biology, but also have the potential to reveal useful targets for intervention. The lack of molecular tools for the functional characterization of genes of interest, however, has hindered progress in this regard. Here we report the development and validation of transfection methodology for this parasite. We first selected from our P. marinus EST collection a highly expressed gene, which we designated "MOE" (PmMOE), to which we fused at the C-terminus the enhanced green fluorescent protein (GFP) as a reporter gene (pPmMOE-GFP). The exogenous DNA was introduced into the trophozoite stage of the parasite by electroporation using the Nucleofector technology. The transfection efficiency was 37.8% with fluorescence detected as early as 14 h after electroporation, with the transfectants still remaining fluorescent after 8 months even in the absence of drug selection. The 5' flanking region was essential for transcription; constructs with 100 and 204 bp flanking the transcription start site also drove transcription effectively. Polymerase chain reaction (PCR) and Southern blot analyses was consistent with integration by non-homologous recombination. This transfection technique, the first one reported for a member of the Perkinsozoa, provides a new tool for studies of gene regulation and expression, protein targeting, and protein-protein interactions, and should significantly contribute to gain further insight into the biology of Perkinsus spp.
Fernández-Robledo
José A
JA
University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 701 E. Pratt Street, Suite 236, Baltimore, MD 21202-3101, USA. robledo@umbi.umd.edu
Lin
Zhuoer
Z
Vasta
Gerardo R
GR
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D056890:Eukaryota / Q000235:genetics*; D005453:Fluorescence; D017930:Genes, Reporter; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010274:Parasitology / Q000379:methods*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014162:Transfection
D016054:DNA, Protozoan; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins