Biolistic transformation of Schistosoma mansoni: Studies with modified reporter-gene constructs containing regulatory regions of protease genes.
Biolistics of the flatworm parasite Schistosoma mansoni facilitates the accurate spatial expression of transgenes under the control of gene-specific promoter elements. To improve transgene expression, either in the number of positive worms and/or an increased transgene signal per worm, we tested plasmid constructs incorporating 5' and 3' gene-specific genomic fragments, and parts of the open reading frame for two S. mansoni proteases, cathepsins F and D (SmCF and SmCD). GFP-expression was gut-localized, a novel finding for SmCD and consistent with previous data for SmCF. The mCherry fluorescent protein can also operate as a reporter. Though certain constructs imparted stronger and better distributed signals per positive worm, the low yields throughout (1-5% positive per experiment) precluded further quantifications of improvement. Electroporation of the same constructs was also weakly efficient (1-10% positives per experiment). However, reporter signals were found in tissues other than the gut, which may represent dysregulated transcription.
Dvorák
Jan
J
Sandler Center for Basic Research in Parasitic Diseases, California Institute for Quantitative Biosciences (QB3), 1700 4th St., University of California, San Francisco, CA 94158-2550, USA. hdvorak76@centrum.cz
Beckmann
Svenja
S
Lim
Kee-Chong
KC
Engel
Juan C
JC
Grevelding
Christoph G
CG
McKerrow
James H
JH
Caffrey
Conor R
CR
D000818:Animals; D019470:Biolistics; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism; D056646:Cathepsin F / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012550:Schistosoma mansoni / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D015801:Helminth Proteins; D010447:Peptide Hydrolases; D056646:Cathepsin F; D002402:Cathepsin D