Aguirre-Arteta, Ana Maria: REGULATION OF DNA METHYLATION DURING DEVELOPMENT: ALTERNATIVE ISOFORMS OF DNA METHYLTRANSFERASE

Kapitel 2. Materials and Methods

2.1 RNA ISOLATION

Total RNA extracted from mouse organs (strain C57Bl6) and cultured muscle cells (C2C12) was used as a template in RT reactions. The animals were killed by cervical dislocation and the organs were immediately taken, rinsed briefly in PBS and either frozen in liquid nitrogen or in isopentane precooled in liquid nitrogen depending on the subsequent applications, respectively RT or in situ hybridization.

During the RNA work a series of precautions were taken to avoid RNase contamination. Glassware was baked at 200oC for at least 4 hours and water was treated with DMPC (Dimethylpyrocarbonate). A 10% DMPC stock was prepared in ethanol, which was then diluted 1:100 in double distilled water, left at room temperature overnight and finally autoclaved.

Total RNA extractions were done with a Qiagen kit (RNeasy) following the instructions of the manufacturer. The purity of total RNA was checked by running ~ 1µl of total RNA on a 1% agarose gel made in TAE buffer (0.04 M Tris-acetate/ 0.001 M EDTA pH 8) with 0.5 µg/ml ethidium bromide. The two ribosomal RNA bands (23S and 18S) were clearly detected when the gel was viewed under ultraviolet light (UV) and smears (as indicator of RNA degradation) were not seen between the two bands. To quantify the total RNA an aliquot was measured by UV absorbance at 260 nm (A260) and 280 nm (A280) where the absorbance of 1 in a 1 cm path length corresponds to a RNA concentration of 40 µg/ml. The absorbance ratio of 260 nm and 280 nm gave an estimate of the purity of the solution. Pure RNA solutions had A260/A280 values between 1.7-2.

2.2 REVERSE TRANSCRIPTION

To assay for Dnmt1 gene expression, total RNA from mouse tissues and cells was reverse transcribed into cDNA. For this reaction hexamer primers of random sequence as well as oligonucleotides of defined sequence were used. Random hexamers are used when a particular mRNA is difficult to copy in its entirety, because of the presence of sequences that cause the RT to stop synthesis. With this method, all RNAs in a population serve as templates for first-strand cDNA synthesis. Most specific is to use an oligonucleotide containing sequence information that is complementary to the desired target RNA. The advantage of using a specific primer is that only the desired cDNA is produced, facilitating the subsequent PCR amplification. The following components were mixed in a total volume of 20 µl to perform a reverse transcription reaction: 1 µg of total RNA was mixed with 1 x reaction buffer (100 mM Tris/ 500 mM KCl pH 8.3), 5 mM MgCl2, 2-10 pmol of primer, 1 mM each dNTP and 20 units of avian myeloblastosis virus (AMV) reverse transcriptase enzyme (Roche Molecular Biochemicals). The mixture was then briefly vortexed and incubated at 25oC for 10 min to allow the primer to anneal to the RNA, followed by incubation at 42oC for 1 h during which the RNA was reverse transcribed resulting in cDNA synthesis. The AMV enzyme was then denatured by incubating the reaction at 99oC for 5 min and then the mixture was cooled down to 4oC for 5 min.


14

2.3 POLYMERASE CHAIN REACTION

PCR was used to amplify segments of DNA that were between two regions of known sequence using either genomic or cDNA as a template. Genomic DNA was prepared from mouse embryos as follows: i) the sample of interest was cut into small pieces using a blade and making sure that the sample stayed frozen at all times by keeping it in liquid nitrogen; ii) the pieces of sample were placed in an eppendorf tube and 850 µl of buffer (10 mM Tris pH 8.5/ 5 mM EDTA pH 8/ 0.2% SDS/ 200 mM NaCl) were added containing proteinase K at a final concentration of 150 µg/ml; iii) the tubes were placed at 50oC for 5 h or at 37oC overnight in a shaking water bath and from time to time the samples were vortexed to disrupt the tissue; iv) the samples were then heated at 95oC for 10 min.

For the PCR reactions , either 5 µl from the RT product (~0.25 µg of cDNA) or 100 ng genomic DNA were used in a total volume of 50 µl containing 1 x PCR buffer (100 mM Tris/ 50 mM MgCl2/ 500 mM KCl pH 8.3), 0.2 mM each dNTP, 15 pmol from each primer and 5 units of Taq polymerase. Different Taq enzymes were used depending on the size of the fragment expected: for up to 2000 bp, the Taq polymerase was used; for bigger fragments, the Long Expand PCR kit (Roche Molecular Biochemicals), which contains a mixture of a thermostable Taq and Pwo DNA polymerases with a 3‘ to 5‘ proofreading activity, was used. For the amplification of cDNA, oligos located in cDNA areas spanning intron locations were used to control for possible genomic DNA contamination.

The PCR mixtures containing all the necessary reagents were denatured by heating at 94oC, then cooled to a temperature which allows the oligonucleotide primers to anneal to their target sequences (usually 5oC less than the primer‘s melting temperature), finally the annealed primers were extended with DNA polymerase. The cycle of denaturation, annealing and DNA synthesis was repeated for ~ 30 cycles, so that the products of one round of amplification served as templates for the next, doubling the amount of desired DNA product in each round. The reactions were carried out on a DNA Thermal Cycler (Biometra) and when optimization of annealing temperatures was necessary the Mastercycler Gradient (Eppendorf) was used. The latter allows the use of a gradient function which enables changes of temperature between 4oC and 99oC to be tested simultaneously.

The amplified DNA fragment was separated by gel electrophoresis on a 1% agarose gel in TAE buffer containing 0.5 µg/ml ethidium bromide. The fluorescence from the DNA bands with the intercalating dye was visualized by exposing the gel to UV light. Depending upon the expected size, the following DNA molecular weight markers were used: SmartLadder (Eurogentec), 100 bp Ladder, Lambda DNA cut with BstEII or Lambda DNA cut with Hind III (all from New England BioLabs). Comparison of the fluorescence intensity of the PCR fragments with the standard bands from the molecular weight marker provided, in addition, an estimation of the amount of DNA in the samples.

Fresh PCR products of the desired size were gel purified with Qiaex II (Qiagen). The gel purification method is based in the adsorption of DNA molecules to silica particles in the presence of high salt. After washing away the non-nucleic acid impurities, the DNA was eluted in ~ 20 µl of 10 mM Tris-HCl pH 8.5. Fresh PCR products were also directly ligated into the appropriate vector depending on the subsequent applications (pCRII vector for sequencing or in vitro transcription, pCR3.1 vector for protein expression in mammalian cells, both vectors are from Invitrogen).


15

2.4 PLASMID DNA TRANSFORMATION INTO ESCHERICHIA COLI AND SELECTION OF RECOMBINANTS

The plasmid DNA was transferred into bacteria by a process known as transformation, in which the plasmid is taken up by bacteria that had been pretreated to make some of the cells temporary permeable to small DNA molecules, i.e., competent . The new phenotype conferred by the plasmid (i.e., resistance to an antibiotic) allows selection of bacteria that had been successfully transformed. After transformation, the Escherichia coli cells were plated on Luria Bertani (LB) medium with the appropiate antibiotic and incubated overnight at 37oC. The next day, isolated bacterial colonies were checked either by PCR using oligos located in the vector and insert or by small scale plasmid DNA isolation using the alkaline lysis method and subsequent restriction enzyme analysis. Briefly, bacteria were lysed by treatment with a solution containing SDS (denatures bacterial proteins) and NaOH (denatures chromosomal and plasmid DNA). The mixture was neutralized with potassium acetate, causing the plasmid DNA to reanneal rapidly. Most of the chromosomal DNA and bacterial proteins precipitated, as well as the SDS which formed a complex with potassium, and were removed by centrifugation. The reannealed plasmid DNA was then concentrated by ethanol precipitation. Restriction enzyme and/or PCR analyses of the plasmid DNA allowed identification of the plasmids containing the right insert, i.e., of the correct recombinants. Selected recombinant bacteria were grown in large scale and subsequent isolation of plasmid DNA was carried out with the Plasmid Maxi kit following the instructions of the manufacturer (Qiagen). Briefly, this method is based on a modified alkaline lysis procedure, followed by binding of plasmid DNA to an anion-exchange resin under appropriate low salt and pH conditions. RNA, proteins and low molecular weight impurities were removed by a medium salt wash. Plasmid DNA was eluted in a high salt buffer, concentrated and desalted by isopropanol precipitation.

2.5 SEQUENCING

Plasmids were sequenced to verify that they contained the desired inserts and to control for unwanted mutations. Samples were processed by the dye terminator method (Applied Biosystems) and purified from unintercalated labeled dNTPs by Centri-Sep spin columns (Applied Biosystems). The samples were then mixed with 4 µl buffer (deionized formamide and 25 mM EDTA pH 8 containing 50 mg/ml Blue dextran in a ratio 5:1 formamide:EDTA-Blue dextran), denatured by heating at 94oC for 4 min, and loaded onto a sequencing gel on an automated DNA sequencer (Applied Biosystems). This sequencing method is based on the dideoxy method developed by Sanger . Briefly, this technique involves the synthesis of a DNA strand from a single-stranded template by DNA polymerase and depends on the fact that dideoxynucleotides (ddNTPs) are incorporated into the growing strand in the same way as the conventional deoxynucleotides (dNTPs). However, ddNTPs differ from dNTPs because they lack the 3‘-OH group necessary for chain elongation. With the dye terminator only molecules that have terminated are labeled, and the four base-specific terminations are carried out in the same tube and then analyzed in the same lane of a gel. The latter is possible because a different fluorescent dye is attached to each nucleotide and incorporated into the DNA chain during the copying reaction catalyzed by DNA polymerase. The samples were then separated on a polyacrylamide gel and the fluorescent nucleotides excited with a laser beam. The emitted fluorescence was collected by detectors and sent to a computer, where the appropriate software converted the data into nucleotide sequence. For


16

sequence assembling, editing and alignments, the Lasergene software programs Edit Seq, SeqMan and MegAlign (DNASTAR, Madison) were used. Some samples were sequenced by the companies GATC or MWG.

2.6 NORTHERN HYBRIDIZATION

This technique was used to study the number, size and levels of Dnmt1 transcripts in different tissues and it is based on the hybridization of a labeled nucleic acid probe (RNA or DNA) to a complementary sequence of mRNA.

2.6.1 Probe labeling

cDNA fragments subcloned into pCRII vectors were prepared by restriction digestion of plasmids and isolated by gel purification. For radioactive labeling of DNA fragments the random primer extension method (Amersham) was used and the enzyme required for this process was Escherichia coli DNA polymerase I (Klenow fragment). The procedure relies on the ability of random oligonucleotides to anneal at multiple sites along the length of a DNA template. Starting from these oligonucleotides annealed to the DNA, the DNA polymerase I synthesizes new complementary strands incorporating labeled dNTPs present in the reaction mixture.

The reaction mixture contained: 25 ng of DNA (which have been previously gel purified) and 5 µl of random nonamer primers (Amersham), which were brought to 50 µl final reaction volume with double distilled water. This initial mixture was denatured by boiling for 5 min. Keeping the tube at room temperature, the unlabeled dNTPs (0.1 mM each, omitting those to be used as label) and reaction buffer (from Amersham, containing Tris-HCl pH 7.5, 2-mercaptoethanol and MgCl2) followed by the radiolabeled dNTPs and DNA polymerase I (Klenow fragment) enzyme (0.04 units/µl) were added. The reaction was then incubated at 37oC for 1 h. Phosphorus-labeled dNTPs (3000 Ci/mmol [alpha-32P] dCTP and 3000 Ci/mmol [alpha-32P] dATP) were used. Non incorporated labeled nucleotides were removed from the probe by precipitation with 0.1 volume of 4 M LiCl and 2.5 volumes of chilled absolute ethanol and incubation at -70oC for 30 min and centrifugation at 13000 g for 15 min at 4oC. The pellet was then washed with ~ 100 µl of cold 70% ethanol, air dried and dissolved in 20 µl of double distilled water. The efficiency of the labeling reaction was monitored by a scintillation counter (Beckman).

2.6.2 Hybridization

Hybridization and washing conditions varied depending on the stringency required to obtain the highest signal and the lowest background. The hybridization solution is one of the critical parameters to consider and should contain blocking reagents such as herring sperm DNA, as blocking reagent, to prevent nonspecific binding of RNA probes to the blotting membrane. The hybridization temperature also needs to be optimized to ensure optimal hybridization to the target and minimal nonspecific binding of the probe. The final, stringent wash is usually 10oC below or in some cases the same as the hybridization temperature. The buffer used was from Clontech or according to Church et al. (7% SDS/ 1% BSA/ 0.25 M NaHPO4 pH 7.2/ 1 mM EDTA pH 8). Prehybridization which contained only the hybridization buffer (5 ml) without the labeled


17

probe was done for 4 h and hybridization overnight, containing 5 ml hybridization buffer and 2 x 106 cpm/ml labeled probe. The probe and 50 µg/ ml of herring sperm DNA were denatured by boiling for 5 min before adding to the blots. The temperature for pre- and hybridization was 68oC. After hybridization the blots were washed twice 20 min with 0.1x SSC, 0.1% SDS at 50oC with continuous shaking. The blot was retrieved from the hybridization bottles and the excess of wash solution was removed not allowing the membrane to dry. The blot was covered with plastic wrap and exposed to a phosphorimager screen (Molecular Dynamics) for 24 h to one week. The signals were detected by scanning the screens on a phosphorimager using the Image Quant software (Molecular Dynamics). The probe was removed from the blot by incubating for 10 min in boiling distilled water. This process was repeated three times and the water allowed to cool for 10 min before the blot was stored in plastic wrap at -20oC, until needed for new hybridization experiments.

2.7 IN SITU HYBRIDIZATION

The principle of in situ hybridization is very similar to Northern hybridization and is based on the annealing of a labeled nucleic acid probe (RNA or DNA) to a complementary sequence of mRNA. The two techniques differ in that the starting material for a Northern is a tissue extract, whereas the primary material for in situ hybridization is a histological tissue section. The relative cell position in the tissue is lost with Northern blots and mRNA levels are averaged from all the cells contained in the original sample. However, in situ hybridization can detect amounts of mRNA contained in a single cell. Furthermore, since in situ hybridization is a histological technique, tissue structure is preserved and it is possible to precisely identify the cell type expressing the gene of interest. A major advantage of in situ hybridization is that it allows the maximal use of tissues that may be in short supply while an organ extract might yield sufficient RNA for one or two Northern blots only. These Northern blots are useful to quantify specific mRNAs, but large amounts of tissue extracts are required. Although Northern blots can be probed multiple times for different mRNAs, in fact this is limited to four or five different probes at most, since repeated stripping of the blots leads to RNA loss from the membranes. In situ hybridization is a semi-quantitative technique but provides information concerning the cellular source of the RNA.

There are many different ways to do in situ hybridization and it can be done with radioactive or non-radioactive probes. The use of 35S labeled riboprobes to hybridize frozen tissue sections has been proven to be the most sensitive method . 32P labeled probes used for Northern blots are not suitable for the in situ technique due to the high energy produced by 32P, which does not allow the detailed cellular resolution of the signal. To control for genomic DNA hybridization and unspecific binding, concurrently with the slides containing the antisense probe, adjacent tissue sections were also included containing a control sense RNA probe.

2.7.1 PROBE LABELLING

For riboprobe preparation 1 µg of plasmid DNA containing T7, SP6 or T3 RNA polymerase binding sites was linearized by digestion with a restriction enzyme downstream (antisense probe) or upstream of the insert sequence (control sense probe). The linearized DNA template was then purified by two phenol extractions (Phenol/Chloroform/Isoamylalcohol: 25/24/1) and LiCl/ethanol precipitation. The DNA was transcribed at 37oC in 20 µl total volume for 2 h with 2 µl 1x transcription buffer


18

(Promega) (200 mM Tris-HCl pH 7.5/ 30 mM MgCl2/ 10 mM spermidine/ 50mM NaCl), 2 µl 10mM GTP/ATP, 2.5 µl each alpha-35S CTP (800 Ci/mmol), alpha-35S UTP (800 Ci/mmol) and 1 µl of the appropriate RNA polymerase enzyme (20 units). The labeled probe was recovered by LiCl/ethanol precipitation and washed with ethanol as described in preparation of probes for Northern hybridization (section 2.6.1). A sense probe, was also labeled as a negative control.

Long probes penetrate less effectively into fixed tissue and the magnitude of this effect depends on the tissue preparation and type of probe used. To increase penetration of the probe in the tissue, long fragments used as RNA probes (~ 1000 nucleotides) were reduced to an average of 150 to 200 nucleotides by alkaline hydrolysis. The alkaline hydrolysis was carried out by dissolving the probe in 50 µl of DMPC treated water and the pH adjusted to 10.2 by addition of 30 µl of 0.2 M Na2CO3 and 20 µl of 0.2 M NaHCO3 and incubated at 60oC for 30 min. The hydrolysis was stopped by addition of 3 µl of 3 M sodium acetate pH 6 and 5 µl of 10% glacial acetic acid. The hydrolyzed probe was precipitated with ethanol and resuspended in a small volume of DMPC water.

2.7.2 SAMPLE PREPARATION

Tissue sectioning

Handling of glass, reagents and the extraction of organs from the animal was as in section 2.1. If the tissue was dense and compact (e.g. muscle), the specimen was frozen in isopentane precooled in liquid nitrogen. Isopentane shortened the time needed for freezing. Fragile tissues were placed in a plastic capsule containing a freezing agent (Tissue Tek), before being frozen. Frozen tissues were sectioned with a cryotome (Microm) into 6-12 µm thick slices, thaw-mounted at 42oC on coated slides (Superfrost/Plus, Fisher Scientific) and immediately re-frozen by placing the slides in the plastic slide boxes kept in the cryostat or on dry-ice and stored at -70oC until use. The frozen tissue slides were first left at room temperature for 1 h to thaw before tissue sectioning.

Fixation

Unlike blotting membranes, tissues are not chemically uniform and respond differently to fixation protocols. Procedures that preserve good morphology and retain RNA usually do not give high hybridization efficiency. Extensive fixation preserves histological detail and prevents loss of target RNA during hybridization and wash procedures. However, milder fixation increases accessibility of the hybridization probes to the RNA target (and to any other molecules such as antibodies) and reduces the possibility of chemical modification of the target RNA and concomitant loss of hybridization. Thus it is important to optimize fixation and prehybridization treatments of tissue to find the best compromise between these requirements. Cross-linking fixatives were favored because they provided much better retention of cellular RNA and help to inactivate nucleases in tissues.

The frozen tissue sections were fixed for 20 min in 4% paraformaldehyde (8 g of paraformaldehyde were dissolved in 200 ml PBS by adding 1-2 pellets of NaOH, the pH was then adjusted to 7 with HCl). The sections were then washed three times 3 min in PBS.


19

Acetylation

The purpose of this step was to cover the section with negative charges to repel nucleic acids and thus reduce electrostatic binding of the probe to the section. The tissue slides were incubated for 10 min in the acetic anhydride solution (250 ml distilled water/ 3.1 ml Triethanolamine/ 675 µl acetic anhydride). After the incubation period the slides were washed 3 min in PBS.

Dehydration

The sections were dehydrated by passing the slides through an ascending ethanol series (50%, 70%, 90%, 96%) and were then left to dry at room temperature for 2-3 h.

2.7.3 HYBRIDIZATION

The slides were placed in a box with a piece of filter paper saturated with buffer (4 x PBS/50% formamide) during the prehybridization and hybridization steps. The prehybridization step was carried out by covering each slide with 100 µl of hybridization buffer (50% deionized formamide/ 10 mM Tris-HCl pH 7.5/ 5 mM EDTA pH 8/ 2 x SSC/ 150 µg/ml herring sperm DNA/ 150 µg/ml yeast tRNA/ 10% dextran sulfate/ 1 x Denhardt´s solution/ 0.1 mM UTP/0.1 mM CTP) without probe and incubating at 37oC for 4 h. Hybridization was performed with a mixture containing the hybridization buffer and the 35S-labeled riboprobe (108 cpm/µl). This mixture was heated at 95oC for 10 min and placed on ice. DTT was then added at a final concentration of 0.1 M. The probe mixture was vortexed and 10 µl was added to the sections. Siliconized coverslips were placed on top of each sample and incubation was done overnight at 42oC. The coverslips were removed from the sections by soaking the slides in 1x SSC.

After hybridization the sections were washed 20 min at 60oC in 5 x SSC/ 1 mM DTT, 30 min at 60 oC in 2 x SSC / 50% formamide/ 1mM DTT, 2 x 10 min at 37oC in RNase-buffer (10 mM Tris-HCl pH 8/ 1mM EDTA pH 8/ 0.5 M NaCl), 30 min at 37oC RNaseA digest (10 mg/200 ml) to remove nonspecifically bound single-stranded RNA probe, thus reducing the background signal in autoradiographs , 10 min at 37oC in RNase buffer, 1h at 60oC in 2 x SSC, 10 min at 60oC in 0.1 x SSC.

The slides were dehydrated by dipping them for a few minutes in an ascending ethanol series and were left to dry at room temperature. The slides were exposed to a Phosphorimager screen to produce a fast low resolution image or dipped in photographic emulsion for high cellular resolution images. The latter involved dipping of slides into a nuclear track emulsion (Amersham), whereby the sections are covered with a thin layer of the emulsion (silver bromide crystals in gelatin). The emulsion was first melted at 42oC and after dipping the slides in it, these were left to dry at room temperature for 1 h and then exposed in the dark at 4oC. Exposure times varied from 1 to 4 weeks depending on the probe and sample. To avoid artifacts, the slides were allowed to warm up to room temperature before photographic development. Decay of radionucleotides leads to the formation of latent images in the emulsion layer. Latent images were converted into real images by photographic development (Kodak) and fixation (Kodak). The slides were then counterstained with hematoxylin (Sigma).


20

2.8 CELL CULTURE AND TRANSFECTION

The cells used were mouse C2C12 myoblasts and COS-7 cells (African green monkey kidney fibroblast-like cells transformed with SV40T antigen; ). The C2C12 cells were used because they are a well characterized tissue culture skeletal muscle differentiation system to screen for Dnmt1 isoforms during myogenesis. These cells were studied at different stages of growth and differentiation (see Results section). The COS-7 cells were used to test the translation efficiency of Dnmt1 isoforms by immunofluorescence and Western blot analyses (see Results section).

Both types of cells were grown in a humidified atmosphere of 5% CO2 and at a temperature of 37oC. The cells were maintained at subconfluent densities in growth medium containing Dulbecco´s modified Eagle´s medium (DMEM) with 10% and 20% fetal bovine serum (FBS) for COS and C2C12 cells respectively, and were split every 48-72 h. Proteolytic enzymes such as trypsin in combination with EDTA were used to detach the cells from the growth surface to harvest or to subculture them. Frozen cells were thawed by placing the vials in a 37oC water bath by gently agitating the vial, cell suspension was then pipetted into a plate containing pre-warmed growth medium. For long term storage cells were harvested as above and washed once with complete medium. The cells were then resuspended in complete medium and counted. The cells were stored in complete medium with 10% DMSO and aliquots of cells (about 106 cells/ml/cryovial) were frozen and kept in liquid nitrogen. C2C12 cells were transferred to DMEM with 5% horse serum (differentiation media) whenever differentiation was needed.

Transient transfections were done with the corresponding eukaryotic expression plasmid in COS-7 cells by the lipofection method (GenePorter), following the instructions of the manufacturer (Gene Therapy Systems, Inc.). Seventy two hours after transfection, cells were washed twice with PBS and either fixed with 3.7% formaldehyde in phosphate buffered saline and immunostained or pelleted for subsequent cell extract preparation and Western blot analysis.

2.9 IMMUNOFLUORESCENCE ANALYSIS

2.9.1 TISSUE SECTIONS

Organs were collected from the animal as in section 2.1 and fixed overnight with Serra‘s (60% absolute ethanol/ 30% formaldehyde/ 10% glacial acetic acid) or Carnoy‘s solution (60% absolute ethanol/ 30% chloroform/ 10% glacial acetic acid). Both have proven to give equally good results. The next day the organs were embedded in paraffin with a Techno-Tec1 automat (Pathotec).

The sectioning of paraffin embedded material was done with a rotary microtome (Microm). After each cut the section was floated on a thermostatically controlled water bath (40-45oC). Then a slide was dipped under the section and upon lifting the section adhered to the slide. The slides were then left to dry on a warming plate.

Sections were deparaffinized by incubating at 60oC for 1 hour, then in Xylol for 15 min, in isopropanol for 5 min, in a descendent ethanol series (96%, 70%, 50%) 5 min each and finally in PBS for 5 min. The samples were then placed in 0.01 M citrate buffer pH 6 and autoclaved for 20 min at 94oC.


21

2.9.2 CULTURED CELLS

Cells were treated were treated with 3.7% formaldehyde in PBS for 10 min which leads to the formation of chemical cross links between free amino groups. Fixation with formaldehyde does not allow access of the antibody to the specimen and therefore was followed by a permeabilization step using a nonionic detergent. After fixation the coverslips were washed twice with PBS. Permeabilization of the fixed cells was performed by incubation in 0.2% Triton X-100 in PBS for 5 min at room temperature. The cells were finally rinsed and kept in PBS.

2.9.3 IMMUNOSTAINING

Fixed samples (tissues or cells) were blocked for 30 min with 0.2% fish skin gelatin. After fixation, incubation with the first antibody (Ab) for 2 hours was done. The unbound antibody was removed by three 10 min washes with PBS-Tween-20 (0.2%). The secondary fluorescently conjugated Ab was then added and incubated for 1 h. After washing as before, nuclear DNA was stained for 5 min with Hoechst 33258 at a concentration of 1 µg/ml. Finally, samples were washed again and mounted in Mowiol, which hardens within a few hours forming a permanent preparation.

Secondary antibodies with different fluorochromes (fluorescein or rhodamine) were used. Under the appropriate illumination, fluorochromes are excitated alllowing the localization of the antibodies and, through them, the distribution of the antigen under study. The methods used are indirect detection and relied on a labeled secondary antibody that recognizes specifically the unlabeled primary antibody.

Protein expression and subcellular localization was also analyzed by epitope tagging. The tag itself is a small open reading frame, and fusion of tags with the protein under study was achieved by standard molecular biology methods. The epitope tag is a short amino acid sequence, that is placed within the coding region of the target protein to specify the binding site for a known monoclonal or polyclonal antibody. The tagged target protein can then easily be detected using well characterized antibodies directed to the tag. Tagged proteins were then produced in COS-7 cells using an appropriate expression vector (pCR3.1 from Invitrogen). The expression and localization of tagged proteins were monitored in transfected COS-7 cells. The Flag tag is not derived from any known protein and contains an 8-amino acid peptide (Asp, Tyr, Lys, Asp, Asp, Asp, Asp, Lys) that is hydrophilic and can be placed at both the amino and carboxyl termini of the target protein. There are two commercially available antibodies, M1 and M2, that recognize the Flag tag. The M1 antibody requires the amino terminus of the epitope to be available, thus the tag is placed at the amino terminus of the protein. The M2 antibody was the one used, which recognizes the same Flag epitope when placed internally within a protein or at the carboxyl terminus.

2.10 MICROSCOPY

Immunofluorescence samples were examined and photographed on a Zeiss Axioplan 2 microscope equipped with phase-contrast and epifluorescence optics, using 63x Plan-Apochromat, 2.5x, 20x, 40x Plan-Neofluar and 4x Achroplan objectives.

A Zeiss AxioLab microscope equipped with phase-contrast and darkfield condenser, was used to examine in situ hybridization specimens using 4x, 10x, 20x and 40x Achroplan


22

and 2.5x Plan-Neofluar objectives.

Images were taken with CCD cameras (SensiCam, PCO and ProgRes, Kontron Elektronik) using Zeiss Axiovision and Photoshop software.

2.11 CELL EXTRACTS AND WESTERN BLOT ANALYSIS

Transfected cells were harvested by centrifugation at 13,000 x rpm for 5 min and cell pellets were resuspended in ice-cold RIPA buffer (500 mM Tris-HCl pH 8/ 150 mM NaCl/ 1% NP-40/ 0.5% DOC/ 0.1% SDS) with protease inhibitors (leupeptin, aprotinin, pepstatin). The cell suspension was incubated on ice for 5 min and extracted proteins were recovered from the supernatant after centrifugation at 13000 x rpm for 5 min. The protein extracts were denatured by boiling in Laemmli sample buffer (2% SDS/ 20% glycerol/ 250 mM Tris-HCl pH 6.8/ 10% beta-mercaptoethanol/ 0.1% bromophenol blue) prior to loading onto a gel. Proteins were separated on a 10-20% gradient SDS polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (BIO-RAD) with transfer buffer (48 mM Tris/ 39 mM glycine/ 0.037% SDS and 20% absolute methanol) using a semidry blotter (Hoefer, 1h at 60 mA). The PVDF membrane was blocked by treatment with 5% nonfat milk powder in PBS (blocking buffer) for 30 min and then incubated in a solution of the primary antibody diluted with blocking buffer and 0.2% Tween 20 for 2 h at room temperature. The blot was washed in PBS containing 0.2% Tween 20 three times 10 min, incubated with the secondary antibody for 1 h and finally with Streptavidin-HRP for half an hour. The signals were detected with the ECL+ reagent according to the instructions of the manufacturer (Amersham). Primary and secondary antibodies were used at the following dilutions: 1:5,000 anti-FLAG antibody M2 (Kodak); 1:10,000 anti-mouse IgG biotin (Amersham); 1:500 Streptavidin-HRP (Amersham). The signals were recorded on a luminescent image reader (Fuji). Images were assembled and annotated using Adobe Photoshop and Adobe Illustrator on a Power Macintosh computer.


© Die inhaltliche Zusammenstellung und Aufmachung dieser Publikation sowie die elektronische Verarbeitung sind urheberrechtlich geschützt. Jede Verwertung, die nicht ausdrücklich vom Urheberrechtsgesetz zugelassen ist, bedarf der vorherigen Zustimmung. Das gilt insbesondere für die Vervielfältigung, die Bearbeitung und Einspeicherung und Verarbeitung in elektronische Systeme.

DiML DTD Version 2.0
Zertifizierter Dokumentenserver
der Humboldt-Universität zu Berlin
HTML - Version erstellt am:
Tue Sep 17 12:11:03 2002