Detection by in situ polymerase chain reaction of a channel catfish gene within cells and nuclei

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An in situ polymerase chain reaction (ISPCR) procedure was developed for analysis of single-locus genes of the channel catfish (Ictalurus punctatus). Intact leukocytes, suspended in phosphate buffered saline (1.0 x 106 cells/milliliter), were distributed on microscope slides and digested with proteinase K (6 μg/ml) for 1 h at 27°C. The PCR was performed on the slides with a thermal cycler (PTC-100, MJ Research, Inc., Watertown, MA). Primers were designed to target the gene Ig H, encoding the constant region of the immunoglobulin heavy chain. After amplification with biotin-labeled primers, the intracellular PCR products were detected by color development or fluorescent methods. The cells on treatment slides were strongly positive for the target gene. This was verified by use of an indirect procedure, in which the ISPCR was performed using unlabeled primers, followed by use of a biotin- labeled probe. The ISPCR technique was also applied to the location of the gene in interphase nuclei. Two copies of the target gene were revealed in positive interphase nuclei. For validation of the ISPCR technique, we performed the technique on a mixture of channel catfish leukocytes and fetal donkey dermal cells. More than 90% of the catfish cells exhibited fluorescence associated with the presence of the target gene, whereas no fluorescence was observed on the fetal donkey dermal cells. Liquid-phase PCR was also included as a positive control in each experiment and yielded products of the expected size (303 base pairs) and DNA sequence. Localization of a single-locus gene by ISPCR is new to nonmammalian species and will provide an important tool for gene mapping and transgenic studies in fishes.

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Applied Immunohistochemistry

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