Escherichia coli K12 DH5α

Hanahan, 1983; Bethesda Research laboratories, 1986

Escherichia coli K12 Sure

Stratagene, La Jolla, CA, USA

Escherichia coli K12 XL1-Blue

Bullock et al., 1987; Stratagene, La Jolla, CA, USA

Escherichia coli K12 Top10

Invitrogen, Leek, Netherlands

Escherichia coli K12 Invα F`

Invitrogen, Leek, Netherlands

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.

pCR2.1, TA cloning kit

Invitrogen, Leek, Netherlands

pGEM, TA cloning kit

Promega, Mannheim

RNaseA

Boehringer Mannheim, Mannheim

ProteinaseK

Boehringer Mannheim, Mannheim

T4 DNA-ligase

Boehringer Mannheim, Mannheim,

Promega , Mannheim

Taq-DNA-polymerase

Boehringer Mannheim, Mannheim

Trypsin

Biochrom, Berlin

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

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

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 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.

PCR conditions:

concentration of Mg2+

2mM

concentration of dNTPs

0.4mM

94 oC

1 min

98 oC

15 sec

66 oC

20 sec

30x

72 oC

1 min

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 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

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round)

Conditions for bisulphite PCRs

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

30 x

95 oC

1 min

59 oC

90 sec

58 oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

peg3 amplification

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round)

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

61 oC

90 sec

30 x

61 oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

Igf2 DMR2 amplification

Buffer:

10 x PCR buffer (25 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round)

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

54 oC

90 sec

30 x

60 oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

H19 amplification (3’ part of the upstream DMR)

Buffer:

Boehringer No. 2 (Long distance PCR kit, 22,5 mM MgCl2)

Primers:

F1, R1 (1st round)

F1, R2 (2nd round)

PCR conditions:

1st round PCR

2nd roundPCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

57oC

90 sec

30 x

57 oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

H19 amplification (5’ part of the upstream DMR)

Buffer:

Boehringer No.2 (Long distance PCR kit, 22,5 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R1 (2nd round)

PCR conditions:

1st round PCR

2nd roundPCR

95 oC

5 min

95 oC

5 min

95oC

45 sec

95 oC

45 sec

65oC

1 min 15 sec

30 x

62 oC

1 min 15 sec

30 x

72 oC

1 min 45 sec

72 oC

1 min 45 sec

72 oC

10 min

72 oC

10 min

Snrpn DMR1 amplification

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

oC

90 sec

30 x

oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

Snrpn DMR2 amplification

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R1 (2nd round)

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

59 oC

90 sec

30 x

57 oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

Xist amplification

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round)

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

61 oC

90 sec

35 x

59 oC

90 sec

35 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

α-actin amplification

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round)

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

57 oC

2 min

5 x

55 oC

2 min

5 x

72 oC

3 min

72 oC

3 min

95 oC

30 sec

95 oC

30 sec

57 oC

2 min

25 x

55 oC

2 min

25 x

72 oC

90 sec

72 oC

90 sec

72 oC

5 min

72 oC

5 min

mylC amplification

Buffer:

10 x PCR buffer (15 mM MgCl2)

Primers:

F1, R1 (1st round)

F2, R2 (2nd round)

PCR conditions:

1st round PCR

2nd round PCR

95 oC

5 min

95 oC

5 min

95 oC

1 min

95 oC

1 min

53 oC

90 sec

30 x

52 oC

90 sec

30 x

72 oC

90 sec

72 oC

90 sec

72 oC

10 min

72 oC

10 min

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)

10 x PCR buffer (15 mM MgCl2)

60 μl

dNTPs (10 mM )

60 μl

primers * (forward + reverse,10μM each)

12 + 12 μl

Taq (4 U / μl)

6 μl

formamide (ultra-pure)

12 μl

dd H2O

438 μl

* . 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:

95oC

5 min

95 oC

10 sec

65 oC

30 sec

35 x

72 oC

1 min

72 oC

5 min

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).