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Non-radioactive labels in transgene detection

was next used to transform 40 pl of XL1-Blue MRF’ Kan supcrcompetent E. coli cells. Whitc (putative positivcs) transformants were used for "miniprep" plasmid purification according to Lech and Brent (1989). In order to select for the clones carrying the 187 bp insert, the "miniprep" plasmids were hydrolyzed using Ksp I and Dra II restriction enzymes. The plasmids with a digestion fragment of 305 bp (187 pb of specific insert + 118 pb of the multiple cloning site) were amplified with the specific primers of v-Ha-ras, and a representati ve positi ve plasmid (showing an amplified product of 187 bp) was used for further sequencing.

Non-radioactive sequencing of recombinant plasmids was carried out following the protocol of the Di%™Taq DNA sequencing kit (Boehringer Mannheim, Meylan, France). In short, aftcr alkaline denaturation of 1 pg of plasmid, 2 pmol of the M13/pUC sequencing primer 5’-labelled with digoxigenin (5’-GTAAAACGACGGCCAGT) was used for the annealing reaction at 55°C for 10 min. After addition of 3 U of 7a<7polymerase, theextension termination was realized at 70°C. Following standard electrophoresis, the sequencing gel was- blotted onto a Nylon™ membranę (Boehringer Mannheim, Meylan, France) by capillarity and the sequencing products were UV-fixed onto the membranę. The membranę was then ineubated in presence of alkaline phosphatase/anti-digoxigenin antibodies conjugate using as a chemiluminescent substrate, AMPPD™ (10 pg/ml, Tropix, Bedford, MA, USA), before being exposed to XAR™ films (Kodak, Rochester, N Y, USA) for2 hrs. Following the chemiluminescent reaction, a colorigenic revelation was achieved using 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as described by Rihn et al. (1995a).

In silu Hybridization

Buffered formalin tissues were deparaffined with xylenc, washed in 100% ethanol and air dried. The labelled probc (v-Ha-rassense or antisense primers; see section "Labelling and Validation of Probcs") was added (200 ng/slide) to 50 pl deionized formamide, 30 pl of 25% (w/v) dextran sulfate and 10 pl 20 x SSC for hybridization in a humid chamber at 37°C for 2 hrs. The slides were then washed twice in 2 x SSC at room temperaturę for 10 min. and twice in 0.2 x SSC for the same time. The detection was based on the use of antidigoxigenin antibody-alkalinc phosphatase conjugate (1/ 1000) in 0.1 M Tris*HCl, 0,1 M NaCl and 0.05 M MgCl₂ (pH 9.5) to which NBT/BCIP had been added.

mRNA Expression in Organs obtained with various Fixatives Mammary tumors and kidneys were fixcd in three different fixatives Solutions: i) Bouin’s; ii) Clarke’s solution (alcohol/ acetic acid; 2/1 v/v) and iii) 4% buffered paraformaldehyde. Five tissue sections (7 pm) were stirred with 1 ml of iso-octane according to the method described by Stanta and Schneider (1991). After three extractions, the pellet was washed twice with 0.5 ml ethanol and finally dried under vacuum. The pellet was suspended in 200 pl of 1 M guanidium thiocyanate, 25 mM 2-P-mercaptoethanol, 0.5% lauroyl sarcosine, proteinase K 1.2 mg, 50 mM Tris«HCl (pH 7.5) and ineubated at 45°C for 6 hrs.

Briefly, following a phenol/chloroform extraction, the nucleic acids were precipitated in isopropanol using 4 pg of glycogen, suspended in 40 pl finał volume containing 50 mM Tris»HCl, 50mMKCl,8mMMgCl₂,1 mMdNTP, lOmMdithiothreitol, 10 mM random hexamers, RNasin™ inhibitor (Sigma, St. Louis, MI, US A)40 U and DNase l-RNase firee (EC 3.1.21.1.), pH 8.3. DNA was hydrolyzed for 1 hr. at 37°C. Following DNasel inactivation (lOmin. at95°C), 20 pl of the hydrolysate were used for cDNA transcription with 50 U of M-MuLV reverse transcriptase (EC 2.7.7.49.) at 37°C for 1 hr. while the remaining 20 pl were used for negative control. The amplification was carried out in 200 pl finał volume with the sense and antisense primers (10 pmol each) of v-Ha-r<w defined above using the same buffer as for retrotranscription, 5 U of Taq polymcrase and 8 mM KC1. The cycler program was 35 times: 40 sec. at 60°C, 30 sec. at 72°C, 40 sec. at 92°C. The samples were electrophoresed in TBE-agarose gels, transfenred to a nylon membranę and, following a hybridization with 20 ng of PCR-labelled probc (see below), the specific DNA was re vealed using a chemiluminescent DIG™ revelation previously described (Rihn et al., 1995b).

Labelling and Validation of Probes The oligonucleotide probes (200 pmol) used for in situ studies were end-labelled with a mixture of dATP (0.5 mM) and digoxigenin-dUTP (0.05 mM) with 2.5 U of terminal transferase (EC 2.7.7.31.) in the reaction buffer (200 mM potassium cacodylate, 25 mM Tris*HCl, 0.25 mg/ml BSA, 5 mM CoCl₂, pH 6.5). The reaction was ineubated at 37°C and the labelled probe was precipitated with 0.1 vol. of 4 M LiCl and 3.0 vol. of prechilled ethanol in the presence of 20 pg of glycogen and 10 mM of EDTA. Following precipitation, the obtained pellet was dried and suspended in TE buffer.

The amplified v-Ha-ray probes were randomly labelled using the same PCR procedurę as above, but replacing 2.0 mM dTTP by 1.3 mM dTTP + 0.7 mM digoxigenin-dUTP in order to obtain digoxigenin-labellcd probes (Lanzillo, 1990). The labelled probes were precipitated ovemight at -20°C with 2.0 pl of 0.5 M EDTA, 6.2 pl of 4 M LiCl and 150 pl of absolute ethanol. The pellets were washed twice with a 20% (v/v) aqueous ethanol solution and dried under vacuum using an Alpha 1 -4™ freeze dryer and an Alpha R VC rotational* vacuum-concentrator (Christ, Ostcrode/Harz, Germany).

Following quantification by UV absorbance (1 unit A_260nm = 37 pg single-strand DNA and 1 unit A_260nm = 50 pg of doublc-strand DNA), the labelled probes were serially diluted in 100 pl of 10 mM Tris*HCl, EDTA 1 mM (pH 8.0) denatured by adding 100 pl of 0.4 N NaOH, 25 mM EDTA for 5 min., deposited onto Nytran NI2™ membranes using a Minifold apparatus (Schlcisher and Schuell, Darmstatt, Germany). After further neutralization using 3 M NaCl, 0.5 M Tris»HCl (pH 7.8), the membranę was equilibrated in 6 x SSC solution and the probes were crosslinked onto the membranę by UV (UV Stratalinker™ 1800, 1.20 mJ/cm² of membranę) (Stratagene, La Jolla, CA, USA).

Membranę blocking and colorigenic probe detection were

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