Optimized in situ PCR method for the detection of gene transfer vector in histological sections

Background Detection of transferred genes in histological sections has been problematic due to low transfection efficiency and copy number achieved wi th current vectors. In situ polymerase chain reaction (in situ PCR) is a ne w method for the detection of low-abundance nucleic acid targets in tissue sections. Methods We have adapted in situ PCR method for the detection and histologic al localization of transgene DNA after in vivo and ex vivo retroviral gene transfer by using mild fixation and permeabilization methods. We used 4% pa raformaldehyde/15% sucrose fixation combined with proteinase K permeabiliza tion and microwave treatment. PCR signal was detected with non-radioactive digoxigenin-dUTP tailed oligonucleotide sense-probe. Results The method was applicable for both paraffin-embedded and frozen tis sue sections and reached the sensitivity to detect a few copies of target D NA sequence per cell. Conclusions In situ PCR is a sensitive method to localize integrated gene t ransfer vectors and to analyze the relationship between expression of the t reatment gene and biological effects in the transfected tissues. Copyright (C) 2001. John Wiley & Sons, Ltd.