A simplified whole mount in situ hybridization protocol using DIG-labelled DNA probes to visualize gene activity in Heterodera schachtii.
Information concerning gene expression during the nematode's life cycle is rapidly accumulating as a result of different screening approaches. In the majority of the cases, the initial characterization of these genes involves determination of their temporal and tissue-specific expression patterns. This preliminary insight into the characteristics of newly isolated genes allows the formulation of a hypothesis and sets the course for further research. Here, we present an optimised method to visualize gene expression in the beet cyst nematode Heterodera schachtii by means of whole mount in situ hybridization. Two different probes for targets with known expression pattern in other nematode species were used to optimise the protocol. It was experimentally observed that the use of vacuum-infiltration during fixation resulted in a fast and complete penetration of the fixative, which was essential to preserve the morphological constitution of the nematode tissue. Some other modifications were introduced that significantly reduced the experimental time without loss of efficiency. As such, we were able to localize the expression pattern of some novel genes with a possible function in the pathogenesis of this nematode.
Vanholme
Bartel
B
Vakgroep Moleculaire Biotechnologie, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Universiteit Gent, Coupure Links 653, B-9000 Gent, Belgium.
Tytgat
Tom
T
De Meutter
Jan
J
Gheysen
Greetje
G
Vanhoutte
Isabelle
I
Coomans
August
A
Gheysen
Godelieve
G
D000818:Animals; D001937:Brassica / Q000469:parasitology; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D004076:Digoxigenin / Q000737:chemistry*; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization / Q000379:methods*; D009348:Nematoda / Q000235:genetics* / Q000254:growth & development
D015342:DNA Probes; D004076:Digoxigenin