Real-time PCR quantification of the in vitro effects of crustacean immunostimulants on gene expression in lobster (Homarus gammarus) granular haemocytes.
This paper presents data from an investigation of the mode of action of five different crustacean immunostimulants presented to European lobster (Homarus gammarus) granulocytes cultured in vitro. The experiments were designed to test whether or not the immunostimulants could cause the short-term up-regulation of genes coding for immune proteins without causing the cells to degranulate. Quantitative measurements of mRNA transcript abundance were made using real-time PCR and it was first necessary to isolate the complete gene sequences coding for the proteins prophenoloxidase (proPO), beta-1,3-glucan binding protein (betaGBP) and beta-actin (beta-act) in the lobster. These sequences were used to design TaqMan primer and fluorescent probe sets. The presented data indicated that the majority of the tested immunostimulants did not induce the up-regulation of immune-related gene expression in the granulocytes in isolation. Alternative modes of action, including the in vivo up-regulation of gene expression in haemopoetic tissues, are discussed.
Comparative Immunology Group, Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, Scotland, UK. ch40@st-andrews.ac.uk
Chris
C
Hauton
John A
JA
Hammond
Valerie J
VJ
Smith
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D004156:Catechol Oxidase / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006434:Hemocytes / Q000187:drug effects* / Q000378:metabolism; D037102:Lectins; D008297:Male; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D000199:Actins; D000276:Adjuvants, Immunologic; D002352:Carrier Proteins; D004792:Enzyme Precursors; D037102:Lectins; C037937:glucan-binding proteins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase