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carried out using the procedurę described above.

Evaluation of the Transgene Copy-Number Two primers which match specifically at their 3’-OH end wiih the v-Ha-ras gene were used in combination with one primer set which specifically amplifies the c-Ha-ras gene (one primer matching perfectly in 3'-OH, and one primer matching an intronic sequence lacking in the v-Ha-raj gene). Both sets of primers were used in the same PCR™ reaction and yielded an amplification (as described in the "mRNA Expression in mammary Tumors" section) of 187 bp and 207 bp DNA fragments for the v-Ha-raj and c-Ha-rar genes, respecti vely. Following this step, the PCR products were serially diluted (1/2 to l/32),electrophoresed, transferredontoa nitrocellulose membranę (Stratagene), and hybridized using the v-Ha-ras probe labelled with Dig-d-UTP™. Colorigenic revclation was performed as described by Rihn etal. (1995a). Immersion of the membranes in a toluene bath renders them transparent, and they were then fixed with Scotfs concentrate (Surgipath™, Labonord, Villeneuve d’Ascq, France) on a glass slide in order to allow the quantification of the colored bands using a scanner spectrophotometer (Dual Wavelength Flying Spot Scanner CS-9000™, Shimadzu, Kyoto, Japan), that employs a software program FDU-3™ (Shimadzu). The scanner densitometeroperated in absorbance/transmission modę, ata wavelength of 550 nm (reference wavelength of420 nm) and a beam size of 2.0 x 0.4 mm². The recorded parameters were: ordinate from -0.1 to 2.0 absorbance units, abscissa in mm corresponding to the optical course of the beam for all peaks.

RESULTS AND DISCUSSION

Following a period of 2 to 6 months, transgenic v-Ha-ras mice (Oncomice Neo 01) spontaneously develop mammary adenocarcinomas, as well as Harderian adenomas and lymphomas. The transgene expression was initialy determined by in situ hybridization using a tailed antisense probe. The label efficiency of the 20 mer-tailed probe was measured as indicated in the "Labelling and Validation of Probes" section: a dilution corresponding to 5 fmol of the labelled probe gave a marked signal using colorigenic detection (Fig. 1A).

As shown in fig. 2A an unambiguous signal was detected by in situ hybridization in the mammary adenocarcinoma HP4884 at a magnitude of 400. mRNA of v-Ha-ras were not detected using a sense oligonucleotide probe (Fig. 2B). Using the NBT/ BCIP revelation protocol (Fig. 2A), as well as the

Fast Red™ (Fig. 2C), the coloration seemed located in the nuclear compartment of the celi. In order to verify this observation, the slides were examined at a magnification of 1000 times (Fig. 2D). This fig. shows intense nuclear and nucleolar coloration with NBT/BCIP indicating the presence of v-Ha-raspre-mRNA. This is not surprising sińce Ha-ras mRNAs have a very short life in the cytoplasm and are rapidly degraded (Barbacid, 1987; Łowy and Willumsen, 1993), their nuclear and nucleolar localizations indicate that pre-mRNA are processed in the nuclear matrix and are localized in spliceosomes (Lawrence et al., 1989), and the transgene construct favors extremly elevated synthesis of v-Ha-ras mRNA in the organs under steroid hormone control such as the mammary gland (Sinn et al., 1987).

Indeed, the mutated v-Ha-ras gene is actively expressed due to the presence of the glucocorticoid response element (GRE, an enhancer seąuence) and the TATAA box of the MMTV in the 5’ regulatory region of the transgene. Nevertheless, it is not possible to assign the signal obtained in the mammary gland to the proto-oncogene (c-Ha-ras) or the transgene (v-Ha-ras) mRNA on the basis of in situ hybridization using a common probe for the detection of v-Ha-ras as well as c-Ha-ras mRNAs.

Fig. 1 Probe validation. A) Slot-dot of serial dilutions of the tailed oligonucleotide 4212 (from 5 pmol to 5 fmol); B) Slot-dot of serial dilutions of the PCR-labelled v-Ha-ra5 fragment (187 bp, from 500 fmol to 50 amol). The labeling reaction was achieved as indicated in the section "Labelling and Validation of Probes”.

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