Construction of subtracted EST and normalised cDNA libraries from liver of chemical-exposed three-spined stickleback (Gasterosteus aculeatus) containing pollutant-responsive genes as a resource for transcriptome analysis.
The three-spined stickleback (Gasterosteus aculeatus) is ideally suited to laboratory studies, while its wide distribution in the northern hemisphere gives it great potential as a sentinel organism. In the setting of a UK-wide collaboration (Fish Toxicogenomics) we have developed a microarray for transcriptomic analysis of chemical responses in populations of G. aculeatus under laboratory and field conditions. Although several EST libraries are available for this species none are from chemical-exposed fish and thus unlikely to include a full set of pollutant-responsive genes. To harvest such transcripts cDNA libraries were produced from liver of chemical-exposed mature males. Two normalised full-length libraries were generated by different methods: (1) partial subtraction of polyA+ RNA against solid-phase cDNA using magnetic bead technology; (2) degradation of double stranded cDNA formed by abundant transcripts. To enrich for pollutant-responsive genes a subtracted EST library was also generated. For each library approximately 1.5K clones were sequenced and characterised using Blast2GO. All libraries contained pollutant-responsive transcripts not previously available while additionally the subtracted library was generally enriched approximately 1.2-10-fold for transcripts expected to be induced in response to the pollutants.
Brown
Margaret M
MM
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, UK.
Williams
Timothy D
TD
Chipman
J Kevin
JK
Katsiadaki
Ioanna
I
Sanders
Matthew
M
Craft
John A
JA
D000818:Animals; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D015723:Gene Library; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D023701:Smegmamorpha / Q000235:genetics*; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D014874:Water Pollutants, Chemical