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Non-radioactive labels in transgene detection 911

Therefore the mRNA of v-Ha-ror were detected using a specific retrotranscription followed by polymerase chain amplification (RT-PCR) technique. As described in the "DNA Extraction and Amplification" section, two primers designed to match at their 3'-OH end with the specific mutations of the y-Ha-ras gene at codon 12 and 59 were used in order to amplify only the v-Ha-ras mRNA. Fig. 3 shows the expression of v-Ha-ras mRNA (cDNA) in various organs of transgenic mice such as the kidney, mammary tumor and the thymus (result not shown). The cDNAs of the biot were revealed with an homologous probe labelled according to the PCR protocol described in the "Labelling and Validation of Probes". The label was efficient as 50 amol of the 187 bp fragment gave a elear signal on the dot-blot of the probe validation (Fig. IB).

A representative sample of v-Ha-ras cDNA was used for cloning in pBluescript II SK(+) vector as described in the "DNA Cloning and Sequencing" section. The sequence obtained using the labelled Dig™ sense primer is shown in fig. 4A. As expected, the sequence was typical of a y-Ha-ras cDNA with the specific transition of G to A in codon 12 (mutation: gly—>arg). As has been described elsewhere (Rihn et al., 1995a), the same biot can be submitted to a second revelation using a chemiluminescent substrate for the phosphatase conjugate (namely AMPPD™) following the NBT/ BCIP coloration shown in fig. 4A. Both revelations of a given part of the same vector sequence are illustrated in fig. 4B. It can here be seen that a similar band intensity is revealed for the dideoxy-terminated fragments. Contrary to many

Fig. 3    Southern biot of amplifted v-Ha-ras cDNA following RT-PCR; exemple of the tissues of animal HP 4745.

lane A: kidney cDNA (HP4745, Bouin’s fixative); lane B: mammary tumor cDNA (HP4745, Bouin’s fixative); lane C: kidney cDNA (HP 4745, Clarke’s fixati ve); lane D: mammary tumor cDNA (HP 4745, Clarke’s fixative); lane E: kidney cDNA (HP 4745, buffered formalin fixative), lane F: mammary tumor cDNA (HP 4745, buffered formalin fixative); (+) and (-) with and without reverse transcriptase. Lanes A to F, chemiluminescent detection. The procedurę is described in the "mRNA Expression in Organs obtained from various Fixatives” section.

It is noteworthy that regardless of the fixati ve solution used (Bouin’s, Clarke’s and buffered formalin Solutions), aclear signal appeared following mRNA extraction. As for in situ hybridization, the mRNA were also still detectable using a 4-hrs. fixation in either of the three buffers tested in this work, even if it is thought that Bouin’ s solution causes crosslinking between proteins and DNA, and prevents DNA amplification (Nuovo, 1992). In contrast, buffered formalin does not alterthe DNA molecule. However, in our study, only short fragments of well expressed mRN As were retrotranscribed, allowing an efficient mRNA recovery whatever the fixative used.

observations (see Discussion in Rihn etal., 1995a), the chemiluminescence (Fig. 4B) based method did not improve the colorigenic signal recorded on the biot (Fig. 4A). Chemiluminescence did not improve the sensitivity of the detection, but did yield a faster revelation; two hrs. instead of an ovemight-period essential for obtaining a elear colorigenic signal or an radioautographic revelation when a radiolabelled nucleotide is used.

For the determination of copy-number of the trangene per genome-equivalent, a specific set of primers was designed for both y-Ha-ras transgene and c-

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