Chemiluminescence-based method for genotyping Tg.AC responder mice.

The Tg.AC transgenic mouse model (2) is under evaluation by the National Toxicology Program (NTP) as an adjunct to the conventional two-year bioassay for the identification of chemical carcinogens. The short-term, 26week bioassay relies on the empirical observation that Tg.AC transgenic mice produce skin papillomas when topically treated with chemical carcinogens (1,3). Ongoing studies in our laboratory indicate that a small percentage of Tg.AC mice (<2.0%) can become unresponsive to chemical carcinogens. This unresponsiveness is due to the spontaneous loss of transgene DNA centered at the headto-head juncture of two inverted transgenes. These deletion events are thought to occur during germ line development in both male and female Tg.AC mice. Genomic DNA blots have conclusively linked an altered genotype, produced by spontaneous deletions within a 2000-bp BamHI promoter fragment, to the nonresponsive phenotype (4). Tg.AC mice capable of responding to chemical carcinogens do not contain deletions of this fragment (4). Given the wide use of this model in the pharmaceutical industry and regulatory agencies as an identifier of chemical carcinogens, the need for a non-radioisotopic DNA blot method to identify nonresponding Tg.AC mice is essential. DNA isolation. Using a modified phenol-extraction method, flash-frozen tail tissue (ca. 1.5 cm) were placed in a 1.5-mL microcentrifuge tube containing 570 μL of Tail Lysis Buffer [50 mM Tris-HCl, pH 7.5, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate (SDS)] and 30 μL proteinase K (10 mg/mL) and placed at 55°C overnight. The entire contents of the microcentrifuge tube were poured into a Serum Separation Tube (SST tube; Fisher Scientific, Pittsburgh, PA, USA), and 600 μL of phenol/chloroform/isoamyl alcohol, 25:24:1 (PCI) were added to each sample. The sample was gently mixed and centrifuged for 10 min at 2000× g at 25°C, after which an additional 600 μL of PCI were added, mixed and centrifuged. Chloroform (600 μL) was added, mixed and centrifuged. The top layer (aqueous fraction above the wax plug) was poured into a 2.0-mL microcentrifuge tube containing 200 μL of water saturated with NaCl. The genomic DNA was precipitated by the addition of 1.2 mL of 100% EtOH (mixed by inversion). DNA was removed by spooling onto a sterile gel-loading pipet tip and placed into 200 μL TE (10 mM Tris-acetate, 1 mM EDTA, pH 8.0). The DNA was incubated at 37°C for 15–30 min and gently mixed by hand (do not vortex mix). The DNA concentration and purity were determined by taking spectrophotometric measurements at A260/280. Restriction digest. Genomic DNA (10 μg) was digested using BamH1 (4 U/μg DNA) at 37°C for 18 h. The digested genomic DNA was precipitated by adding 1/10 vol 3 M sodium acetate (pH 5.2) and 2× vol of 100% EtOH. The genomic DNA sample was collected by centrifugation at 14 000× g for 10 min and washed once with 2× vol of 70% EtOH. The genomic DNA sample was dried and redissolved in 17 μL of TE (pH 8.0). Four microliters of loading dye (Amresco, Solon, OH, USA) were added, and the samples were loaded onto a 1.2% agarose gel in 1× TAE (0.04 M Tris-acetate, 0.002 EDTA) and run at 85 V for 4–5 h. The gel was treated with ethidium bromide and photographed for reference. The high-molecular-weight DNA was depurinated by soaking the top portion of the gel in 0.25 N HCl for 10 min. The gel was rinsed in distilled (d)H2O, soaked in denaturing buffer (1.5 M NaCl, 0.5 M NaOH) for 25 min and then soaked in neutralizing buffer (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0) for another 25 min. The DNA was transferred overnight by traditional capillary blotting onto Duralon-UV Membranes (Stratagene, La Jolla, CA, USA) in 20× standard saline citrate (SSC) buffer. The DNA was cross-linked to the membrane by UV exposure and washed with dH2O to remove excess salt and agarose. Fluorescein labeling of DNA. Seventy-five nanograms of the EcoRI/ BamHI zetaglobin promoter fragment of the Tg.AC transgene (1) were labeled by the random primed method in a solution containing fluorescein-11dUTP, supplemented with nucleotides and random nonamer-length primers according to the protocol from Gene Images Labeling Module (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The EcoRI/BamHI zetaglobin promoter fragment in 20 μL dH2O was denatured by boiling for 5 min in a water bath and snap-cooled on ice. Following the manufacturer’s protocol (Catalog No. RPN 3541; Amersham Pharmacia Biotech), the following were added: 10 μL of the nucleotide mixture, 5 μL of the primer mixture, 20 μL of the denatured DNA and dH2O to a total vol of 49 μL, mixed gently in a 1.5-mL tube and placed on ice. One microliter of enzyme solution was then added and gently mixed. The reaction mixture was incubated at 37°C for 1 h. The reaction was terminated by adding 2 μL of 0.5 M EDTA solution, and aliquots were stored at -20°C for up to 6 months without degradation. Hybridization and detection. Hybridization and detection of the fluorescein-labeled probe used in the non-radioisotope genotypic analysis of Tg.AC mice was performed by the protocol and components from Gene Images CDP-Star Detection Module (Catalog No. RPN 3511; Amersham Pharmacia Biotech). Essentially, the membrane was washed for 30 min at 57°C in a prehybridization solution containing 5× concentrated SSC (0.1% SDS), 1/20 vol Liquid Block and 0.5 g/mL dextran sulfate. Seventy-five nanograms of labeled probe were heat-denatured in boiling water for 5 min and then snapcooled on ice. The labeled probe was added to the hybridization buffer and allowed to hybridize overnight at 57°C. The hybridization solution was removed from the blot, then the membrane was washed for 15 min in 1× SSC, 0.1% SDS at 57°C, followed by a second 15-min wash in 0.5× SSC, 0.1% SDS at 57°C. The membrane was incubated at 25°C with gentle agitation in a 1:10 dilution of Liquid Block in Buffer A (100 mM Tris-HCl, 300 mM NaCl, pH 9.5). This was followed by a 1-h incubation in a solution containing antifluorescein-AP conjugate and 0.25 g bovine serum albumin (BSA) in 50 mL Benchmarks