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Non-radioactive labels in transgene detection 913

Ha-ras proto-oncogene as described in the section "DNA Extraction and Amplification". The v-Ha-ras gene was specifically ampłified due to the mismatch of both primers at their 3'-OH end with the cellular Ha-ras proto-oncogene and pseudogene and conversely, the primers specific for the c-Ha-ras gene did not amplify either the v-Ha-ra.y gene or the Ha-ras pseudogene due to their intronic location. Under these conditions, the c-Ha-ras genes and v-Ha-ras transgene gave two products of 207 and 187 bp, respectively, following amplification and hybridization (Fig. 5A). After immersion of the colored biot in a toluene bath and fixation on a glass slide, these bands were scanned using a laser spectrophotometer, and the value of the absorbance at 550 nm was recorded as a function of the shift of the laser beam. The peaks thus obtained are shown in fig. 5B. Whatever the dilution, the absorbance value of the v-Ha-ra.y peak, was two to three fold greater than the value of the c-Ha-ras peak, indicating that this representative transgenic mouse harbored 2 to 3 copies of transgene per genome-equivalent (Table 1). Our experiment shows that colorimetry (namely, relative colorigenic quantitation) is relevant for non-radioacti ve labelling and detection, and provides a numeric-based evaluation of the color density and an analysis without the need for radioactivity. Indeed, in the past, blotted DNA quantitation was only performed with chemiluminescent or radiolabelling detection using luminometers or scintillation counters, respectively(Rohan etal., 1990).Generally,relative quantitative PCR (Q-PCR) was achieved by amplifying together target DNA with an internal control and by comparing the ratio of target DNA versus control DNA (either endogenous or exogenous). This last technique has been used with a temperaturę gradient gel electrophoresis (TGGE) to improve the relative quantitation of the targetDNA(Ferree/a/., 1994; Riesner etal., 1992). The copy-number determination of the transgene is essential for the analysis of copy-number stability in transgenic animals and is therefore a significant part of the validation process which allows one to use transgenic animals for the assessment of Chemical toxicity (Dycaico et al, 1994).

Table 1 Area values of the scanned peaks offig. 5B

Dilution

	1/2	1/4	1/8	1/16	1/32
Arbitrary v-Ha-ras Units c-Ha-ras	184,000 55,800	134,000 24,300*	74,000 44,600	36,000 17,500	10,500 4,400

The values corresponded to the relative units under each area. *This value is probably under estimated.

Fig. 2 In situ hybridization of Ha-ras mRNA; exemple ofthe HP4884 mammary tumor. A) Mammary gland section of an adcnocarcinoma(HP4884); hybridization with the tailed antisenseoligonucieotide 4212;coloration with NBT/ BCIP. a: acinus; B) Same tumor (HP4884); hybridization with the tailed sense oligonucleotide 4206; coloration with NBT/BCIP; a; acinus; C) Same tumor (HP 4884); hybridization with the tailed antisense oligonucleotide 4212; coloration with Fast Red™; D) Same tumor (HP 4884); hybridization with the tailed antisense oligonucleotide 4212; coloration with NBT/BCIP. Magn. A-C) x 400; D) x 1000

Fig. 4    Non-radioactiveseąuences. A)Exampleof acloned v-Ha-rascDNA fragment showing the specific G—>A

transition, colorigenic detection; B) Colorigenic detection of a part of pBluescript II SK(+) using NBT/BCIP (left) and chemiluminescent detection of the same part using AMPPD (right) as described in the "cDNA Cloning and Sequencing“ section.

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