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Optimized in situ PCR method for the detection of gene transfer vector in histological sections
P M Leppänen1, J Koponen, M P Turunen
1A.I. Virtanen Institute, University of Kuopio, Finland.
Background:
Detection of transferred genes in histological sections has been problematic due to low transfection efficiency and copy number achieved with current vectors. In situ polymerase chain reaction (in situ PCR) is a new 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 histological localization of transgene DNA after in vivo and ex vivo retroviral gene transfer by using mild fixation and permeabilization methods. We used 4% paraformaldehyde/15% sucrose fixation combined with proteinase K permeabilization 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 tissue sections and reached the sensitivity to detect a few copies of target DNA sequence per cell.
Conclusions:
In situ PCR is a sensitive method to localize integrated gene transfer vectors and to analyze the relationship between expression of the treatment gene and biological effects in the transfected tissues.