2. Material & Methods

Primordial germ cells for the magnetic bead-based purification were obtained from the mouse embryos of outbred MF1 mouse strain.

The Oct4-GFP transgenic mice were prepared on a mixed F1-129 background. For the isolation of PGCs Oct-GFP males were mated with MF1 females. The mice harbour GFP/Oct4 fusion under the control of Oct4 promoter region lacking the proximal enhancer (GOF18ΔPE - Yeom, et al. (1996), see Fig. 9). This deletion of proximal (but not of the distal) enhancer ensures the germ line specific transgene expression.

	Fig. 9: Scheme of the Oct4/GFP transgene.

Restriction endonucleases were purchased from either Boehringer Mahhneim (Mannheim), New England Biolabs (Beverly,MA,USA) or MBI Fermentas (Vilnius, Lituania).

MiniMacs holder for magnetic bead-based

All other material and equipment were of today laboratory standard.

SOC-medium

SOB-medium substituted with 20 ml 1 M glucose.

For bacterial plates, 15 gr of bacto-agar was added per liter of medium

Bisulphite solution

2,5 M metabisulphite

125 mM hydroquinone

Unless otherwise stated, all the common laboratory solutions were prepared according to Sambrook, et al. (1989).

All primers are listed in 5’ to 3’ orientation.

(as described in Chuma and Nakatsuji (2001))

Ube1XF: TGG TCT GGA CCC AAA CGC TGT CCA CA

Ube1XR: GGC AGC AGC CAT CAC ATA ATC CAG ATG

Colony PCR forward: GCT ATT ACG CCA GCT GGC GAA AGG GGG ATG TG

Colony PCR reverse: CCC CAG GCT TTA CAC TTT ATG CTT CCG GCT CG

Universal sequencing primer (M13 For-40): GTT TTC CCA GTC ACG ACG

Reverse sequencing primer (M13 Rev-28): AGG AAA CAG CTA TGA CCA T

In all the cases of the bisulphite PCR nested or semi-nested approach was chosen. The

Primers for the analysis of Lit1 gene( Acc# AJ271885):

F1: tat tat ttt ggt gtt ggt tat atc ggg tta

R1: att ttt ctt caa cac CCT TCT ttt ccc t

F2: ggg tta taa agt tta ggg gtt TTT AGA tt

R2: aaa ctt ttc tat tca act taa ttc cca ac

Primers for the analysis of peg3 gene (Acc# AF105262):

F1: ttt tta gat ttt gtt tgg ggg ttt tta ata

R1: aat ccc tat cac cta aat aac atc CCT ACA

F2: ttg ata ata gta gtt tga ttg gta ggg tgt

R2: atc tac aac ctt atc aat tac cct taa aaa.

Primers for the analysis of Igf2 DMR2 (Acc# U71085):

(as described in Oswald, et al. (2000))

F1: AAC TAA AAT TAT CTA TCC TAT AAA AC

R1: TTG ATG GAT TTA TAT TGT AGA ATT AT

F2: GGA ATT CCC TAT AAA ACT TCC CAA ACA AAC CTT CAA A

R2: GGA ATT CCT GAT TTA TTG ATG GTT GTT GGA TAT TT

Primers for the analysis of H19 upstream DMR (Acc#AF049091):

5’ part:

(as described in Olek and Walter (1997)– the primer combination F9-R9)

F1: GGA ATT CCT ATA TGG GGA TGG GTG TTT AGA AGG GGA T

R1 (~R9): AAA AAC TAA CAT AAA CCC CTA ACC TCA TAA

F2 (~F9): AAG AAA AAG GTT GGT GAG AAA AAT AGA GAT

3’ part:

(as described in Ueda, et al. (2000))

F1 (~Bis6t0b): AGG GAT TTA TAG GGG TGG TAA

R1 (~Bis7t0): AAA TAC ACA AAT ACC TAA TCC CT

R2 (~Bis7t2): CCT AAA ATA CTC AAA ACT TTA TCA C

Primers for the analysis of Snrpn DMR1(AH007008):

(as described inEl-Maarri, et al. (2001))

F1 (~Bi-MoSN-F1): AAA TTT GTG TGA TGT TTG TAA TTA TTT GGG

R1 (~Bi-MoSN-R1): AAA ATC CAC AAA CCC AAC TAA CCT TCC

F2 (~Bi-MoSN-F2): AAT TAT ATT TAT TAT TTT AGA TTG ATA GTG AT

R2 (~Bi-MoSN-R2): TTT ACA AAT CAC TCC TCA AAA CCA A

Primers for the analysis of Snrpn DMR2:

F1: gtg taa gtt tgg taa aat att at

R1: aat taa aaa aat aaa cca aca ata aca

F2: aaa aaa taa att tct tat act ata aaa c

Primers for the analysis of α-actin gene (Acc# M12347):

(as described in Oswald, et al. (2000))

F1: AAG TAG TGA TTT TTG GTT TAG TAT AGT

R1: ACT CAA TAA CTT TCT TTA CTA AAT CTC CAA A

F2: ggt ttt agt tat ttg ggt tag ggt

R2: CCT ACT ACT CTA ACT CTA CCC TAA ATA

Primers for the analysis of mylC gene (Acc# X12972):

(as described in Oswald, et al. (2000))

F1: GTA TAA TAA ATT TGG ATA GGT AAA GGT TAG

R1: AAA CCT AAA ACA CTA ATC TTA AAA ATT TTA

F2: ATA TTA TAG TAG GGG TTG GAA TGA TTA AAG

R2: CCT ATT AAA CTA ATC TAA AAA ACA ATC CTC

Primers for the analysis of Xist promoter region (Acc# U50909):

F1: tgg ttt gtt taa gta gaa gat ata ttg

R1: aaa aat ctt acc aaa aca tat caa aac

F2: gta tag ata ggt gtg tga ttt aat g

R2: ttt aat ata ttt tct taa ata aac c

The primordial germ cells from 11.5 dpc and 12.5 dpc mouse embryos were isolated using the immunoaffinity purification. For the selection the SSEA1 (Stage Specific Embryonic Antigen 1 – Fox, et al. (1981)) antigen with characteristic germ line specific expression was used. The expression of this marker vanishes around 13.5dpc, thus, using the antibody-based procedure, we obtained only very few PGCs from 13.5dpc embryos suggesting that the obtained fraction was not representative. The samples of 13.5 dpc PGCs were obtained by FACS sorting of the cell suspension obtained from the genital ridges of the Oct4/GFP transgenic mice with germ line specific GFP expression (Yeom, et al. (1996)). For the reason of simplicity, higher yield and purity also the samples of early post-migratory germ cells (10.5 dpc) were obtained using the same procedure.

The whole genital ridges (together with mesonephos) were isolated from 10.5-13.5 dpc mouse embryos and washed in PBS (day 0 refers to the day of a vaginal plug). The tissue was subsequently trypsinised (trypsin:EDTA solution, room temperature) in order to prepare a single cell suspension (clumps were removed by pipetting the suspension up and down several times). The trypsin was neutralised by adding MM medium and the cells recollected by centrifugation (5 minutes, 1 500 rpm). Afterwards, the sample was resuspended in 300 μl MM medium and incubated on a shaker with 50 μl of TG1 (mouse monoclonal anti SSEA1) antibody (Gomperts, et al. (1994))for 45 minutes at 4oC. After changing the medium (addition of 200 μl of MM medium, centrifugation and resuspending in 300 μl of MM medium), the cells were incubated with 20 μl of anti-mouse secondary antibody coupled to magnetic beads (Miltenyi Biotec) for 20 minutes at 4oC on a shaker. In the following step the cell suspension (volume increased to 500 μl) was loaded onto a column on the MiniMACS (Miltenyi Biotec) holder (pre-equilibrated with 500-1000 μl of MM medium) and the negative fraction was collected (this fraction was used as “somatic cells” in the control experiments – see Results). After two washing steps (500 μl of MM medium each), 500 μl of fresh MM medium was added on top of the column detached from the magnetic field and the fraction of the positive cells was collected with the help of a plunger. Finally, the positive cells were washed with PBS.

MM medium:

The isolated genital ridges were trypsinised as described above. The trypsin was neutralised by adding MM medium. After centrifugation (5 minutes, 1 500 rpm) the cells were resuspended in PBS and FACS sorted on a MoFlo (Cytomation Bioinstruments GmbH, Freiburg im Breisgau).

The purity of isolated primordial germ cells was checked using the alkaline phosphatase staining. (The germ cells are characterised by their high level of alkaline phosphatase activity – Ginsburg, et al. (1990)). The aliquot of the isolated germ cells was stained with Sigma Diagnostics Alkaline phosphatase (AP), Leukocyte (no. R86) kit. Shortly, the cells were incubated in staining solution (2,25ml ddH2O, 50 μl sol.1 (sodium nitrate), 50 μl sol.2 (FRV alk.sol.), 50 μl sol.3 (naphtol AS-BI) in dark for 5-10 min and washed in PBS. Cells positive for the alkaline phosphatase activity are stained red.

The purity of isolated fraction always exceeded 95%.

Determination of sex is simple in the case of 12.5 dpc and 13.5 dpc embryos. At this stage of mouse development the genital ridges of male and female show a distinct morphology (see Results Fig. 13). Due to the indistinguishable morphology of genital ridges at earlier developmental stages the sex determination of 11.5 dpc embryos was based on the amplification of Ube1 genes (Chuma and Nakatsuji (2001)). (There are two Ube1 genes in mice, Ube1X on the X chromosome and Ube1Y on the Y chromosome (Imai, et al. (1992)). The primers amplify fragments of both Ube1X and Ube1Y, but the PCR results in products of different size, due to several deleted regions between the two genes. Thus, two distinct bands are amplified from male samples and a single band from female samples.)The procedure was the following: the embryos were decapitated, part of the head tissue boiled in a PCR cycler for 5 minutes and the supernatant used for the sex specific PCR reaction.

The PCR products were subsequently separated on a 2% agarose gel.

For isolation of high molecular weight chromosomal DNA from mouse embryonic tissues the DNEasyTM Tissue Kit (Qiagen) was used. Shortly, the small pieces of tissue were lysed in the ATL buffer in the presence of Proteinase K and incubated at 55oC for 3 hours. After short treatment with Rnase A (5 minutes at room temperature), the samples were mixed with the AL buffer, incubated at 70 oC for 10 minutes and precipitated with ethanol. The entire mixture was subsequently applied onto a DNEasy spin column and centrifuged. After extensive washing with buffers AW1 and AW2, the isolated chromosomal DNA was eluted using 100μl of elution buffer (AE) and stored at 4 oC.

The method of bisulphite sequencing allows the determination of a methylation status of a known DNA sequence. The principle of the method is based on the reaction of single stranded DNA with sodium bisulphite resulting in the conversion of cytosine residues into the uracil residues, while methylated cytosines stay unconverted (for details see 1.7 Molecular techniques used for DNA methylation studies). The bisulphite treated DNA is subsequently amplified in the PCR reaction, the PCR fragments cloned and sequenced.

The modification of a method published by Olek and Walter (1997) is routinely used in our laboratory (Hajkova et al., 2002). To enhance the complete separation of DNA strands, the digested DNA is denatured and embedded into a low melting point agarose. This step ensures the spatial separation of the DNA strands throughout all the following steps.

Two following variation of the procedure was used for the bisulphite treatment of PGCs.

The isolated germ cells were dissolved in 3 μl of PBS and mixed with 8 μl of 2 % hot (80 oC) Sea Plaque (FMC) agarose. The cell/agarose mixture was overlaid with mineral oil and boiled in a water bath for 10 minutes. The samples were immediately transferred into ice and incubated for about 30 minutes to allow the agarose mixture to re-solidify (during the re-solidification an agarose bead containing the cells was formed at the bottom of each test tube). In the following step the agarose beads were overlaid with the 100 μl of lysis solution (solution A: solution B - 1:1, supplemented with 10 μl of Proteinase K (10mg/ml)) and incubated over-night at 50 oC. After several washes with 1 ml of 1x TE solution the agarose beads were equilibrated against the restriction buffer (2 x 15 minutes) and the embedded DNA digested with 20 U of restriction endonuclease over-night at 37 oC (for bisulphite analysis of most of the gene regions described in this thesis the EcoRI restriction endonuclease was used, the only exception was the distal part of the H19 upstream DMR, in which case the PstI restriction endonuclease was used). Following the digestion the agarose beads were denatured with fresh 0,4 M NaOH (2 x 15 minutes), washed for 5 minutes with 0,1 M NaOH, overlaid with 500 μl of mineral oil and boiled for 5 minutes in a water bath. After boiling, the samples were immediately transferred into ice and incubated for about 30 minutes to allow the DNA/agarose mixture to re-solidify. In the next step the agarose beads were overlaid with 500 μl of the bisulphite solution (2,5 M sodium metabisulphite, 125 mM hydroquinone) and incubated in darkness first 30 minutes on ice (the sulphonation step is faster at low temperature) and then for additional 3,5-4 hours at 50 oC (higher temperature is preferred for the deamination step). Following the incubation, the agarose beads were extensively washed with TE buffer (4 x 15 minutes with 1ml of 1 x TE buffer) and treated with fresh 0,4 M NaOH (2 x 20 minutes with 1ml of NaOH solution) (de-sulphonation step). Finally, the agarose beads were washed with TE buffer (2 x 20 minutes with 1ml of 1 x TE) and stored at 4 oC.

Approximately 700 μg of high-molecular -weight DNA was digested over-night in the volume of 21μl with 10U of restriction endonuclease at 37 oC. The restriction mix was subsequently denatured in the boiling water bath for 10 min. After edition of 2M NaOH into the final concentration of 0.3 M, the samples were incubated for additional 15 minutes at 50 oC to assure complete denaturation. The samples were then mixed with 2 volumes of hot 2% LMP agarose and 4-6 10 μl aliquots pipetted into the pre-chilled mineral oil overlaying 750 μl of bisulphite solution (see previous chapter). All the following steps were identical to the procedure described in the previous chapter.

The bisulphite treatment was followed by the gene specific PCR amplification. In all the cases nested (or at least semi-nested) approach was chosen to ensure the highest specificity and sensitivity of the procedure.

Prior to setting up the PCR reaction the bisulphite treated beads were washed twice with dd H2O (2 x 15 minutes with 1 ml of dd H2O) in order to remove the traces of the TE buffer.

If not stated otherwise, the PCR was running in the following conditions:

1 x PCR buffer (15 mM MgCl2 – for the composition see 2.1.5.7. Buffers & solutions), 200 μM dNTPs (each), 20 pmol primer (each), 4 U Taq polymerase. The reaction volumes were the following: 100 μl for the first PCR reaction; 50 μl for the second PCR reaction. One bisulphite treated agarose bead (10 μl) was used as a template for the first PCR, 1-3 μl of the first PCR product were used for setting up the second PCR reaction. All the PCR conditions were optimised using the gradient PCR cycler (Eppendorf) – the conditions with the highest annealing temperature still resulting in a PCR product were chosen for the experiments to ensure the specificity and high rate of bisulphite conversion.

Lit 1 amplification

Conditions for bisulphite PCRs

peg3 amplification

PCR conditions:

Igf2 DMR2 amplification

PCR conditions:

H19 amplification (3’ part of the upstream DMR)

PCR conditions:

H19 amplification (5’ part of the upstream DMR)

PCR conditions:

Snrpn DMR1 amplification

PCR conditions:

Snrpn DMR2 amplification

PCR conditions:

Xist amplification

PCR conditions:

α-actin amplification

PCR conditions:

mylC amplification

PCR conditions:

The products of the bisulphite PCR were separated on the 1 % agarose gel (run in TBE buffer); the specific PCR products were cut out using a sterile scalpel. The DNA was subsequently extracted using the QiaEx II kit (Qiagen). Briefly, the agarose was dissolved in QX1 buffer at 50 oC in the presence of glass milk, the mixture was centrifuged and the glass milk pellet containing bound DNA washed twice with PEbuffer. The pellet was subsequently air-dried for 15 minutes at RT and the bound DNA eluted with 20 μl of dd H2O. The rests of glass milk inhibiting the following ligation were removed by short additional centrifugation.

In all the PCR experiments the unmodified Taq polymerase was used. Since this enzyme is prone to non-template addition of adenine nucleotide(s) to the 3’- terminus of newly synthesised DNA strands a very simple cloning procedure based on those “sticky ends” could be used. The purified PCR fragments were ligated using the TA cloning kit (Promega) into a pGEM based plasmid vector with T-overhangs on both 3’- termini. (In the original experiments the PCR fragments were cloned into a pBluescript based pCR 2.1 TA cloning system (Invitrogen). This procedure, however, resulted several times in a bias towards the cloning of unconverted molecules. The system was exchanged for the pGEM-based vectors, in which case the bias has never been observed).

Depending on the yield of the PCR reaction, 3-5 μl of purified PCR product were added to the ligation mix (all the components provided by the kit) and incubated for 1 hour at room temperature (fast ligation scheme using the 2 x ligation buffer). 5 μl of this ligation reaction were subsequently transformed into commercially available ultra-competent E.coli cells (Top10 or INVαF’ strains, both Invitrogen) or to competent E.coliSure cells prepared in our laboratory (see the following chapter).

The transformation into competent E.coli cells was performed using the heat shock protocol. Briefly, the aliquot of frozen competent cells was thawed on ice, mixed with 5 μl of the ligation mix and incubated on ice for additional 30 minutes. The incubation was followed by heat shock performed at 42 oC for exactly 90 seconds. After a short (1-2 minutes) incubation on ice 250 μl of pre-warmed (37 oC ) SOC medium were added and the cell suspension rigorously shaken for 1 hour at 37 oC. Finally, the transformed cells were plated on LB agar plates supplemented with ampicillin as a selection marker, IPTG (20 μl per plate) and X-gal (50 μl per plate) for the blue/white screening of transformed colonies (the insertion of a fragment into the cloning vector results in the disruption of the lacZ ORF, thus the colonies containing the insert appear white in the screening procedure). Plates were incubated at 37 oC overnight.

30 ml of bacterial LB-medium was inoculated with 300 μl of the over-night E.coli culture and incubated until it reached the cell density of OD560=0,4. All the following steps were carried on strictly on ice. The E.coli cells were centrifuged for 10 min at 6000 g, re-suspended in 15 ml of 50 mM CaCl2 and incubated on ice for 15 min. The centrifugation (10 min, 6000 g) was repeated and the pellet re-suspended in 3 ml of the CaCl2 solution. After the addition of glycerol (to the final concentration 15 %, (v/v)), the 100 μl aliquotes of the cell suspension were shock-frozen in liquid nitrogen and stored at –80oC.

The transformation efficiency was estimated by transformation of 1ng of a control pUC19 plasmid and determined as a number of transformed colonies per 1 μg of plasmid DNA.

The experience of our laboratory showed that some of the bisulphite fragments were unstable in bacterial host. For this reason the transformed colonies were always checked for the presence of the insert of the correct size by colony PCR

The procedure was the following: the PCR mix was pipetted into the wells of 96-well MTP. The white transformed colonies were picked by the autoclaved toothpicks and dipped one by one into the wells of MTP. The same toothpick was used to inoculate the field in a grid of a replica agarose plate (colonies growing on this plate could be used when it was necessary to repeat the sequencing procedure).

Colony PCR mix: (for 25 reactions - 30 μl each)

* . The primers for the colony PCR anneal in the polylinker of the cloning vector, outside the annealing positions for the M13 universal and reverse sequencing primers.

Conditions for the colony PCR:

5 μl of the colony PCR products were loaded onto a 1 % agarose gel in order to check the size of the product. The rests of the PCR reactions belonging to the clones with the correct-size PCR product were frozen and handed over to the sequencing facility of the institute. (Routinely 15-20 clones were sequenced per each transformation.)

The colony PCR products were purified using the fully automated magnetic bead system of our sequencing facility. The purified DNA was subsequently used as a template for the Big Dye sequencing procedure and sequenced on the ABI 377 automatic sequencer.

The sequences were checked individually for their quality using Chromas (Microsoft Windows based) software or using the GCG 10.0 Wisconsin Package operating on a Unix interface. With the help of this Unix-based software the sequences of the parallel clones were piled-up and the information about the methylation status of the tested DNA region converted manually into the dot diagrams presented throughout this thesis.

The isolated germ cells on poly-L-lysin coated slides (Sigma) were swollen in 1 % Na citrate hypotonic solution for 5 min. Freshly prepared fixative acetic alcohol (Methanol: glacial acetic acid 3:1) was dropped directly onto the cells and slowly air dried in a humid chamber. The cell preparations were treated with 100 µg/ml RNase A in 2x SSC at 37 °C for 60 min and with 0.01 % pepsin in 10 mM HCl at 37 °C for 10 min, and then dehydrated in an ethanol series (70, 85 and 100 %). The slides were denatured in 70 % formamide, 2x SSC for 1 min at 80 °C and then dehydrated in an ice-cold ethanol series. After brief air-drying, the slides were first incubated with blocking solution (3 % BSA, 0.1 % Tween 20, 4x SSC) in a Coplin jar for 30 min and then with mouse anti-mC antibody (hybridoma supernatant, a kind gift of A. Niveleau), diluted 1:50 with PBS, in a humidified incubator at 37 °C for 30 min. The slides were then washed in PBS three times for 10 min each and incubated for 30 min with fluorescein-isothiocyanate (FITC)-conjugated anti-mouse IgG (Dianova) appropriately diluted with PBS. After three further washes with PBS, the preparations were counterstained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) in 2x SSC for 5 min. The slides were mounted in 90 % glycerol, 0.1 M Tris-HCl, pH 8.0 and 2.3 % 1,4-diazobicyclo-2,2,2-octane. Images were taken with a Zeiss epifluorescence microscope equipped with a thermoelectronically cooled charge-coupled device camera (Photometrics CH250).