Evaluation by southern blot hybridization of DNA administered with a gene carrier to organs

Abstract

The biodistribution of plasmid DNA administered with gene carriers is an important parameter in evaluating the characteristics of gene carriers in vivo and the expected therapeutic effects of transgenes. Radioisotopic labeling of DNA is the most common method for determining biodistribution, and the amount of labeled DNA present in each organ can be measured directly by measuring the radioactivity of the organ. This technique has several advantages, such as the high sensitivity of detection; a wide dynamic range which allows highly accurate quantification; and the simplicity of the procedure, which facilitates the handling of many samples at a time. However, radioisotopic labeling cannot distinguish between intact and degraded DNA in the organs. In addition, radioisotopes have to be used in an authorized location and, due to the half-life of the radioactivity, within a limited time frame. Alternatively, fluorescently labeled DNA can also be used to assess biodistribution. Although fluorescence detection is advantageous for observing cross-sections of organs under the microscope, it is unsuitable for determining the total amount and quality of the DNA in each organ. In order to solve these problems, Southern blotting and non-radio isotope (RI) probe hybridization techniques can be utilized. The technique of Southern blotting, named after its inventor, Edward Southern, allows the size of the particular DNA of interest within the smear of genomic genes in the organs to be identified. Using this technique, information about the quality (intact or degraded) and quantity of the DNA in organs is obtained. Of course, procedures for non-RI Southern blot hybridization are more complicated, and the accuracy and dynamic range of quantification is lower than with the direct RI system.However, a highly sensitive cooled CCD camera (e.g., NightOWL, Berthold Technologies, Bad Wildbad, Germany) that detects the chemical luminescence from hybridized fragments over a wide dynamic range has been developed. This technique allows both the quality and the quantity of DNA to be evaluated and is expected to be a powerful tool for studying gene delivery in vivo. This chapter provides an example of Southern blot hybridization as applied to detecting genes delivered to organs by intravenous administration with gene carriers into mice. Two kinds of gene carriers, a cationic lipid (DOTAP) and a sixthgeneration dendrimer (dendritic poly(l-lysine), are compared with respect to differences in the pharmacokinetics in tumor-bearing mice.