Brugia malayi: transient transfection by microinjection and particle bombardment.
To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.
Higazi
Tarig B
TB
Division of Geographic Medicine, BBRB 203, University of Alabama at Birmingham, 1530 3rd Ave. South, 35294-2170, USA.
Merriweather
Anthony
A
Shu
Limin
L
Davis
Richard
R
Unnasch
Thomas R
TR
D000818:Animals; D001483:Base Sequence; D019470:Biolistics; D017178:Brugia malayi / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D008863:Microspheres; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000235:genetics; D020038:RNA, Spliced Leader / Q000235:genetics; D014162:Transfection / Q000379:methods*
D018509:DNA, Helminth; D008164:Luminescent Proteins; D012341:RNA, Ribosomal, 5S; D020038:RNA, Spliced Leader; D049452:Green Fluorescent Proteins; D008156:Luciferases