Differential Analysis of Active and Inactive Genes in Human Neutrophils by Chromosomal In Situ Hybridization

Abstract

In situ hybridization has been widely used for localization of specific genes in interphase cell nuclei. The sites of signals, however, do not represent the activity of the gene transcription in the nuclei. To differentially localize genes in active and inactive states, the effect of nuclease digestion prior to in situ hybridization using PCR products of DNA fragments from human X-chromosome, as a probe DNA, was examined in female neutrophils. According to the conventional protocol for chromosomal in situ hybridization, we detected two dot signals in the nucleus. However, the use of Ca/Mg-dependent endonuclease, which extracts inactive gene DNA, resulted in the loss of the one signal of the outer region of the nucleus, but not that of the inner one. In contrast, when exogenous DNase I, which extracts active gene DNA, was used in place of Ca/Mg-dependent endonuclease, the inner signal, but not the outer one, was almost lost in neutrophils. Considering that the outer signal represents inactive X-chromosome DNA in the site of female neutrophil nuclei, these results indicate that the use of our modified protocol may enable us to discriminate the signals of active and inactive genes in interphase cells by in situ hybridization.