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 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.
Leppänen
P M
PM
A.I. Virtanen Institute, University of Kuopio, Finland.
Koponen
J
J
Turunen
M P
MP
Pakkanen
T
T
Ylä-Herttuala
S
S
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007150:Immunohistochemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011817:Rabbits; D012680:Sensitivity and Specificity
D017931:DNA Primers