Characterization of RTG-2 fish cell line by random amplified polymorphic DNA.
The increasing presence of genotoxic chemicals in the aquatic environment has led to the development of both in vivo and in vitro assays for target species. The fish population represents an important level of aquatic ecosystems that can be threatened by increased environmental pollution. The authors have studied the DNA pattern of the RTG-2 fish cell line, a fibroblast-like cell line, derived from rainbow trout (Oncorhynchus mikyss), to use this cell line as an in vitro system to study genotoxicity by means of random amplified polymorphic DNA primers (RAPDs). A constant pattern in the DNA band is essential when an organism or cell line is used to detect DNA alterations produced by genotoxic environmental chemicals. DNA fingerprints with RAPDs were obtained for RTG-2 by testing 26 single and 70 pairwise combinations of primers. Different methods of DNA extraction (chelating resin, salting out, and phenolization), the influence of spectrometric measures at 320 nm in the 260/280 quotient to quantify DNA extracts, genomic DNA and primer concentrations, annealing temperatures, and cell line passage were studied in the cell line characterization. RAPD products were identified by agarose gel electrophoresis. The good results obtained should allow the use of this system as a possible tool for detection of the genotoxicity of aquatic pollutants.
Ferrero
M
M
Toxicology Department, I.S. Carlos III, Madrid, Spain.
Castaño
A
A
Gonzalez
A
A
Sanz
F
F
Becerril
C
C
D000818:Animals; D002460:Cell Line; D016172:DNA Fingerprinting; D017931:DNA Primers; D066298:In Vitro Techniques; D009152:Mutagenicity Tests; D017683:Oncorhynchus / Q000235:genetics*; D019105:Random Amplified Polymorphic DNA Technique; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D017931:DNA Primers; D014874:Water Pollutants, Chemical