Localization of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene by in situ PCR in mouse ovary and testis

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To understand better the biological functions of cells and tissues, it is necessary to investigate the localization of a specific gene at a cellular level. Recently, a new sensitive method, in situ PCR that can detect a few copies of genes within a cell by amplifying the target gene has been developed. In the present study, we optimized experimental conditions for the detection of pgk-1 gene by in situ PCR in the sections of mouse ovary and testis. The ovaries and testes from ICR mouse (7 weeks old) were fixed with 4% paraformaldehyde/PBS and embedded in paraffin. Then 5Epm thick serial sections were prepared. The oligo-DNAs corresponding to a part ( 170 bp) of mouse X-linked pgk-1 gene were synthesized as the primers. To label the amplified DNA, Digoxigenin (Dig)-dUTP (5.0 μM) or FluorX-dCTP (0.2 μM) were used. As a negative control, in situ PCR was done omitting Taq DNA polymerase or the primers. When the effects of various concentrations of proteinase K (PK) (0-100 μg/ml) (37℃ 15 min) on a signal/noise (S/N) ratio were examined using an annealing temperature at 56 the best S/N ratio was obtained at 50 μg/ml for ovarian tissues and at 10 μg/ml for testes. As for PCR cycle, 25 cycles were optimal to detect pgk-1 gene in the granulosa cell and spermatogenic cell nuclei. The S/N ratio of FluorX-dCTP was better than that of Dig-dUTP. Using above conditions, we detected one to four pgk-1 spots in the nuclei of granulosa cells and one to two pgk-1 spots in the nuclei of spermatogenic cells. Therefore, in situ PCR was a useful method to detect a single specific gene in the paraffin sections under the optimal conditions.
日本組織細胞化学会の論文

Localization of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene by in situ PCR in mouse ovary and testis

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