Publication Abstract

Isolation of differentially expressed genes in contaminant exposed European flounder (plachtichys flesus) by suppressive subtractive hybridisation

Isolation of differentially expressed genes in contaminant exposed European flounder (plachtichys flesus) by suppressive subtractive hybridisation

Derek L. Sheader, Karl Gensberg, Brett P. Lyons and Kevin Chipman

Differentially expressed sequences were isolated from laboratory and environmentally exposed European flounder (Platichthys flesus) by means of suppressive, subtractive hybridisation (SSH). Commercial, farmed animals were obtained and given single intraperitoneal injections of 5mg/kg benzo-[a]-pyrene (BaP) or 2mg/kg cadmium (Cd) in controlled laboratory experiments. Livers were then removed and snap frozen at 3 days post exposure. Additional liver samples were obtained from animals from two UK estuaries, the heavily polluted Tyne and relatively clean Alde.

Messenger RNA was isolated from each exposure group and its respective control and cDNA produced by SMART-RACE. Specialised adapters were ligated to either the control or exposed cDNA population and both subsequently pooled together. Sequences common to both populations were allowed to hybridise together and the unhybridised, differentially expressed sequences from the adapter-ligated population enriched and isolated by polymerase chain reaction (PCR). Both up and down regulated genes were isolated in separate SSH experiments (forward and back subtraction).

Subtracted cDNA libraries were successfully produced from each exposure group and cloned into the pCR2.1-TOPO vector. This was validated by the PCR amplification of gene fragments of cytochrome P4501A and metallothionein specifically from the BaP and Cd exposure forward subtractions. Clones were isolated from each exposure, 234 clones from the environmental exposure producing 92 distinct sequences, and 384 clones from each compound studied in the laboratory exposures. Approximately 20% of clones could be tentatively identified by homology with database sequences. Full identification will be pursued for those genes for which differential expression can be confirmed by array technology.

Reference:

Derek L. Sheader, Karl Gensberg, Brett P. Lyons and Kevin Chipman. 2003. Isolation of differentially expressed genes in contaminant exposed European flounder (plachtichys flesus) by suppressive subtractive hybridisation. Marine Environmental Research 58 (2-5) 553-557

Derek L. Sheader, Karl Gensberg, Brett P. Lyons* and Kevin Chipman

Marine Environmental Research 58 (2-5) 553-557