BAC library screening
A PCR reaction for the use in PCR-based screening of the Canis familiaris DogBAC library (Schelling et al., 2002) (Institute of Animal Genetics, Nutrition and Housing, University of Berne, Berne, Switzerland) for a BAC clone containing HMGA1 was established using canine genomic DNA derived from blood. The primers A1In5up (5' GGCATCCGGTGAGCAGTG 3') and A1In6lo (5' CAGGCAGAGCACGCAGGAC 3') were designed using GeneBank sequences AY366395 &NW_876254. PCR parameters were: 95°C for 5 min, followed by 30 cycles of 95°C 30 sec, 59.3°C 30 sec, 72°C 30 sec, and a final elongation of 72°C for 10 min. The corresponding 201 bp PCR product was cloned into the pGEM-T Easy vector system (Promega, Mannheim, Germany) and verified by sequencing. The DNA contigs and alignments were done with Lasergene software (DNAstar, Madison, USA) and various sequences from the NCBI database (AY366395, NW_876254). The verified BAC clone MGA 572 P20 K12 RC was used as probe for the following FISH experiments.
1 ml of canine whole blood was incubated for 72 h in Chromosome Medium B (Biochrom, Berlin, Germany). Subsequently, colcemide (0.1 μg/ml) (Biochrom, Berlin, Germany) was added for 2 hours. The cells were centrifuged at 135 × g for 10 min and incubated for 20 min in 0.05 M KCl. Finally the cells were fixed with methanol/glacial acetic acid. This suspension was dropped on ice-cold slides and dried for at least 7 days at 37°C. The chromosomes were stained by GTG banding for karyotype description. Prior to use in FISH investigations, the slides were destained with 70% ethanol.
Fluorescence in situ Hybridization
MGA 572 P20 K12 RC BAC-DNA was digoxigenin labelled (Dig-Nick-Translation-Kit, Roche, Mannheim, Germany). The hybridization mixture contained 200 ng probe, 40 ng ssDNA, 600 ng sonicated dog DNA, 2 × SSC, 2 × SSPE, 50% formamide and 10% dextran sulfate. 50 μl of this mixture were applied to each slide and the cover slips were sealed with rubber cement. Probe and chromosomes were denatured at 75°C on an Eppendorf Thermocycler gradient, using the in situ adapter. Afterwards, the slides were incubated in a moist chamber at 37°C over night. Cover slips were carefully removed and the slides were incubated in 0.1 × SSC at 61°C and 1 × PBS at RT. Slides were then covered with 100 μl non fat dry milk (NFDM) for 20 min. at 37°C in a moist chamber. For signal detection 100 μl NFDM containing 3 μg of Anti-Digoxigenin-Rhodamine, Fab fragments (Roche, Mannheim, Germany), were added to each slide and again incubated for 20 min at 37°C in a moist chamber, followed by washes with 1 × PBS, 3 × 3 min. at RT. Slides were air dried before chromosomes staining was performed with 25 μl of Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA)
Ten well spread metaphases were examined indicating a signal on CFA 12q11 on both chromatids of both chromosomes CFA 12q11 (Fig. 1). The determination of chromosomes follows the nomenclature of the canine karyotype as described previously .
For genomic characterisation of the canine HMGA1 gene the missing parts were amplified by PCR on the screened BAC clone MGA 572 P20 K12 RC. For the missing part 1 a 858 bp fragment (bankit 1078968) was generated with primer pair A1_6640-6997_upa (5'-GGCGCGGCTCCAAGAA-3'), A1_6_lo_2 (5'-CCAACAGAGCCCTGCAAA-3'), a 1879 bp fragment (bankit 1078465 for the missing part 2 was generated by the primer pair A1_8864-10549_upa (5'-GTCTCACCGTCTGGAGAAT-3'), A1_8864-10549_loa (5'-TCACCGGAGGCTGCTT-3') and for the third missing part a 979 bp fragment (bankit 1078536) was generated with primer pair A1_11223-11834_upa (5'-CTGAGCCCATGCCAGATAA-3'), A1_11223-11834_loa (5'-AGAGATCCCTGCCGTAGT-3'). The obtained PCR products were separated on a 1.5% agarose gel, recovered with QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), cloned in pGEM-T Easy vector system (Promega, Mannheim, Germany) and sequenced for verification. The final genomic canine HMGA1 contig and the identity alignments were created with Lasergene software (DNAStar, Madison, USA) with the generated sequences from the cloned cDNAs described previously and various sequences from the NCBI database derived from the canine genome sequencing (AY366394, AY366395, AY366396, NM_001003387, NW_876254).
Genomic DNA was isolated from the collected 55 Dachshunds samples using the QiaAmp kit (QIAGEN, Hilden, Germany). A specific genomic PCR using the primer pair A1In5up (5' GGCATCCGGTGAGCAGTG 3') and A1In6lo (5' CAGGCAGAGCACGCAGGAC 3') was established allowing the amplification of the complete exon 6 and flanking regions of intron 5 and 6, respectively (Figure 3). In detail the PCRs were performed in a 25 μl volume containing 0.5 μM of both primers (MWG Biotech, Martinsried, Germany), 0.1 mM of each dNTP (Invitrogen, Karlsruhe, Germany) 0.6 units Taq-DNA polymerase (Promega, Mannheim, Germany), 1.5 mM MgCl2 (Promega, Mannheim, Germany), PCR buffer (Promega, Mannheim, Germany) and 2.5 μl template DNA, containing averaged 26.5 ng/μl.
After an initial denaturation step of 5 min at 95°C, the amplification followed in 30 cycles (30 sec. at 95°C, 30 sec at 59.3°C and 30 sec at 72°C). To complete, a final elongation step for 10 min. at 72°C completed the process. The obtained PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), directly sequenced by MWG Biotech (Martinsried, Germany), and additionally digested enzymatically with AluI (Fermentas, St. Leon-Rot, Germany). The occurrence of the described SNP creates a new restriction site for the enzyme AluI (5' AG▼CT 3'). Thus, a digestion with AluI cuts the 201 bp PCR product in two fragments of 69 bp and 132 bp, respectively allowing a verification of the sequencing results.
HMGA1 in vivo localisation
For the HMGA1 in vivo localisation the protein coding sequence of the canine HMGA1a was amplified by PCR using primer pair EcoR1_IY-upATG (5'-CGGAATTCCACCATGAGCGAGTCGAGCTCGA-3'), BamH1_IY-loSTOP (5'-CGGGATCCTCACTGCTCCTCTTCGGAGGACT-3'). The obtained PCR products were separated on a 1.5% agarose gel, recovered with QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), ligated into the pEGFP-C1 vector plasmid (BD Bioscience Clontech) and sequenced for verification.
Cells from canine mammary tumour cell line MTH53a were cultivated using medium 199 (Invitrogen, Karlsruhe, Germany) supplemented with 20% FCS, penicillin, and streptomycin. The transfection was performed according to the manufacturer's instructions using 3 μl FugeneHD reagent (Roche, Mannheim, Germany) in 100 μl PBS (without Mg2+) containing 2 μg of recombinant pEGFP-C1-HMGA1a. After treatment, the cells were incubated for 48 hours in the culture media. The uptake and expression of DNA was verified by fluorescence microscopy.