Southern Blot

Southern blot experiments were performed to analyze the status of transgenes in stable cell lines or mice. The technique bases on the protocol established by Edward Southern [130].

Electrophoretically separated restriction fragments of genomic DNA were transferred onto a nylon membrane and hybridized with labeled probes.

3.2.4.3.1 PCR labeling of probes for Southern Blot analysis

Probes for Southern blot analysis (3.2.4.3) were generated by PCR amplification with digoxigenin-labeled dUTPs with the PCR DIG Probe-Synthesis kit according to manufacturer’s instructions. Dot blot analysis and agarose gel electrophoresis was used to verify sufficient incorporation of digoxigenin into the probe. Optimal concentration of probes for hybridization in Southern blot experiments was evaluated empirically.

3.2.4.3.2 Agarose electrophoresis for Southern blot

Genomic DNA (gDNA) was prepared according to the protocol described in section 3.2.2. 10 µg was digested with an appropriate restriction enzyme o.n. DNA was loaded with 1 × GelPilot Loading Dye (Qiagen, Hilden, D) on a 0.5 % TAE-agarose gel and electrophoresis was performed at 80 V for 16 h. The gel was stained afterwards in an ethidium bromide bath and photographed with a fluorescent ruler to determine the running distance of the marker fragments. Subsequently, the gel was destained in water before continuing with capillary transfer.

3.2.4.3.3 Capillary transfer of gDNA

Electrophoretically separated gDNA must be pretreated to be efficiently transferred to a positively charged nylon membrane (Roche). As the target DNA fragments were in general over 10 kb in size, the DNA was depurinated by submerging the gel in 0.25 N HCl until the bromphenol-blue contained in the loading dye turned yellow. Thereby the fragments break into smaller pieces. After a short wash in ddH₂O, DNA fragments were denatured in 0.5 N NaOH/

1.5 M NaCl until the color of bromphenol changed back to blue. The pH of the gel was neutralized by submerging the gel in 1M Tris /1.5 M NaCl (pH 7.4) for 15 min. In the meantime

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the nylon membrane was submerged in ddH₂O and than equilibrated in 20 × SSC. The capillary transfer was set up as depicted in Figure 11.

Figure 11: Upward Capillary transfer modified from Sambrook and Russel [131]

Transfer of the DNA was carried out by capillary forces from 20 × SSC transfer buffer that is drawn from the reservoir through the gel onto the membrane. The paper towels and weight help to maintain a constant stream of buffer.

Capillary transfer was performed at RT o.n. Then, the DNA was crosslinked onto the wet nylon membrane in an UV-linker by 0.125 Joule/cm². Salt crystals from the 20 × SSC were removed by rinsing the membrane with ddH₂O. Thereupon, the DNA was air-dried before continuing with the hybridization procedure.

20 × SSC pH 7.0 3 M NaCl 0.3 M Na-Citrate

3.2.4.3.4 Vacuum transfer

Vacuum transfer of DNA was performed alternatively to capillary transfer. This method is able to create sharper bands and can be accomplished in a shorter time. To this end, the vacuum blotter was set up with agarose gel, nylone membrane and Whatman paper. Each soaked in 20 × SSC according to manufacturer’s instructions. Depurination was performed directly on the vacuum blot, by soaking 0.25 N HCl through the gel while applying constant pressure of 0.2 bar until the indicator bromphenolblue of the loading dye turned yellow. Excess solution was removed before addition of 0.5 N NaOH/ 1.5 M NaCl to denature the genomic DNA until the indicator turned blue. Neutralization solution (1 M Tris /1.5 M NaCl pH 7.4) was added for 10 min. Final transfer of gDNA was achieved by soaking 20 × SSC buffer through the gel for 1.5 h.

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Crosslinking of the DNA and preparation of the nylon membrane was performed as described previously ( see section 3.2.4.3.3).

3.2.4.3.5 Hybridization of probe

To minimize unspecific binding of probe to gDNA, the nylon membrane was pre-hybridized with DIG-Easy Hyb solution. At least 20 ml of the pre-hybridization solution were added to the membrane in roller-bottles. Incubation was performed at a defined hybridization temperature for 2-3 h. The success of a southern blot analysis is strongly dependent on the right hybridization temperature and probe concentration. The latter one was evaluated empirically ― not exceeding 25 ng/ml―. A starting point to find the optimal hybridization conditions can be calculated with following equation:

T_m= 49.82 + 0.41 × % GC - (600/l) T_hyb = T_m- (20 °C to 25 °C)

(with Tm = melting temperatue; Thyb = hybridization temperature, % GC = percent GC-content, l = length of probe)

Table 4: Conditions of Southern blot probes

probe optimal hybridization temperature optimal concentration

S2-luc-dig 37 °C 20 ng/ml

S3-gfpscp-dig 47 °C 15 ng/ml

The probe was first diluted in 50 µl ddH₂O, boiled 5 min at 95 °C, immediately put on ice to achieve single stranded DNA and then added to 20 ml pre-warmed DIG-easy hyb solution.

The probe was carefully mixed in the solution and then added to the membrane. Hybridization was performed for 16 - 24 h. Unbound probe was removed by two washes with a low-stringency buffer ( 2 × SSC, 0.1 % SDS) at 37 °C or RT for 30 min and 10 min respectively, followed by two washes with a high-stringency buffer (0.5 x SSC, 0.1 % SDS) at 65 °C for 10 min each.

3.2.4.3.6 Detection of labeled target DNA

For detection of the labeled target DNA an alkaline phosphatase conjugated anti-DIG-antibody that specifically hydrolyzes the chemiluminescent substrate CDP-star was used allowing detection of emitted light on an X-ray film (Hyperfilm ECL, GE Healthcare). The DIG luminescence detection kit and CDP-Star were used according to manufacturer’s instruction.

41 3.2.4.4 Fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a technique to visualize the locus of a DNA sequence in the genome. Basic steps involve the preparation of metaphase spreads, generation of specific probes and their hybridization to the chromosomes, as well as detection of the probes by immunofluorescence-labeling.

3.2.4.4.1 Preparation of metaphase chromosome spreads

FISH hybridization was performed on condensed metaphase chromosomes. NIH3T3 were split one day before preparation on a 10 cm² plate at a ratio of 1:3. Demecolcin (Sigma-Aldrich) was added to a final concentration of 0.1 µg/ml to the cell culture medium to yield a maximum of metaphase cells. After 2 h incubation found herein to be optimal for NIH3T3, rounded and detached metaphase cells were harvested into a 15 ml falcon tube. After centrifugation at 300 × g for 5 min, cells were washed with pre-warmed PBS (37 °C) and finally resuspended in 0.5 ml PBS. At a minimal vortex speed, 10 ml of 37 °C warm hypotonic solution was added dropwise and cells swelled during the following 15 min incubation in a 37 °C water bath. Than 1 ml fixative was added, the suspension was carefully inverted and incubated at RT for another 15 min. Cells were centrifuged at 300 × g at RT for 5 min, supernatants discarded, carefully resuspended in 1 ml of residual solution and finally resolved in 7 ml fixative. This step was repeated three times at 4 °C. Afterwards, the cells were resuspended in 1 ml fixative and incubated at -20 °C for at least 10 min. Three to four drops of this suspension was dropped from at least 40 cm distance onto ice cold water covered slides that were purified by sonification and pre-chilled to 0 °C. Slides were incubated on a 40 °C hot plate with high humidity and finally dried at RT. After one week of incubation at RT in the dark, metaphase spreads were further processed.

Hypotonic Solution

0.91 % (w/v) tri-sodiumcitrate-dihydrate pre-warmed to 37 °C, freshly prepared

Fixative

3 volumes methanol : 1 volume glacial acid pre-chilled to -20 °C, freshly prepared

3.2.4.4.2 Probe preparation

Efficient labeling of probes is very important for successful FISH analysis to achieve a good signal to noise ratio. Probes were either directly labeled by incorporation of Diethylamincoumarin-dUTP (DEAC-dUTP) or were labeled with digoxigenin-dUTP or biotin-dATP allowing indirect detection via fluorescence coupled antibodies, i.e. dig-Fab or

anti-42

biotin-Fab. The incorporation of the modified nucleotides was performed by PCR or nicktranslation. The optimal length for probes is 300 – 500 bp. Longer PCR fragments were digested with appropriate restriction enzyme or digested with DNase I. Probes were denatured with 1/10 volume of 3 M NaAc, 2.5 volumes of 100 % EtOH and 1 µl salmon sperm DNA, to prevent unspecific binding. The mixture was centrifuged at 20,000 × g for 20 min, supernatant was discarded and the DNA pellet was washed with 150 µl ice-cold 70 % EtOH. After another centrifugation step, the pellet was dried in a vacuum-centrifuge at 65 °C for 5 min. Probes (for one hybridization window with 25 µl) were dissolved in 5 µl 100 % formamide and denatured in a 37 °C waterbath for 30 min. Renaturation of the single stranded probe was inhibited by addition of 5 µl 40 % dextransulfate in 2 × SSC. The mixture was further incubated at 72 °C for 5 min to improve denaturation and subsequently incubated at 37 °C for 30 min. This mix was stored at - 20 °C until use and applied without further treatment.

The commercial available mouse-pancentromeric FISH probe mouse-Pan-Cy3 (BioCat, Heidelberg, D), was used as control and directly applied for hybridization after denaturation at 72 °C for 10 min.

3.2.4.4.3 Hybridization of FISH probes

Before hybridization, metaphase spreads were treated with pepsin and RNAse to remove cell debris. To this end, the previously prepared metaphase slides were washed with 2 × SSC for 5 min at RT. Then 200 µl RNAse solution (0.2 mg/ml RNAse A in 2 × SSC) was added to the coverslide. After 30 min incubation in a dark moist chamber at 37 °C, slides were washed three times with 2 × SSC. For the pepsin digest, prewarmed 50 ml ddH₂O to 37 °C was mixed with 100 µl 5 N HCl and 15 µl 10 % pepsin. Slides were incubated therein for 70 s, to allow the digestion of cytoplasmic proteins without destroying the chromosome structures. Immediately afterwards, slides were washed two times with PBS for 5 min at RT and then with an ascending ethanol row of 70 %, 90 % and 100 % respectively at 4 °C for 3 min each. Subsequently, slides were air-dried in a dark chamber.

To denature the chromosomal DNA, slides were put into a 72 °C hot denaturation solution (70 % Formamide/ 2 × SSC) for 105 s and immediately transferred to -20 °C pre-chilled 70 % EtOH. Slides were dehydrated by putting them into an increasing -20 °C chilled ethanol row for 5 min each (70 %, 90 %, 100 % ethanol). Finally, slides were air-dried in a dark chamber.

For the hybridization, a 10 µl drop of the probe mixture (3.2.4.4.2) was set on the slide and covered by an 18 mm × 18 mm cover slip by sealing the edges with the rubber glue

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Fixogum. Slides were heated to 72 °C for 2 min, then transferred to 37 °C water bath in a closed metal chamber and incubated for 2 - 3 days.

3.2.4.4.4 Fluorescence-Immunodetection

Hardened fixogum was removed and coverslides carefully removed by dipping slides into 2 × SSC 0.2 % Tween-20. All following steps were performed under light protection of the slides.

Slides were washed three times with 2 × SSC 0.2 % Tween-20 at 42 °C for 5 min, three times with 0.75 × SSC at 60 °C for 5min and once again at 42 °C for 5 min with 2 × SSC 0.2 % Tween-20. Unspecific binding sites for antibodies were blocked with 3 % BSA for 30 min at 37 °C. Slides were washed again at 42 °C with 2 × SSC 0.2 % Tween-20 for 5 min.

Immunolabeling of the probes was achieved by adding anti-biotin-Cy3.5 (1 : 3000 in 1 % BSA) binding to the biotin labeled probes and anti-dig-fluorescein antibody (1 : 150 in 1 % BSA). Incubation was performed for 45 min at 37 °C in a humidified chamber. Excessive antibodies were washed away by incubating slides three times at 42 °C in 2 × SSC for 5 min.

DNA was counterstained with 0.05 µg/ml DAPI in 2 × SSC solution for 2 min in a dark moist chamber and washed with ddH₂O for 5 min. Slides were air-dried afterwards. To minimize fading of the fluorescence marker phenylendiamindihydrochlorid was added to the slides and covered by a cover slide.