fragment in exchange with a CAG-eGFP-anillin expression cassette (AseI, HpaI) in a bluntended

Transcription

1 Supplementary Methods Generation of a CAG-eGFP-anillin lentivirus The psico vector which is based on Lentilox37 8 was truncated via excision of an XbaI-XhoI fragment in exchange with a CAG-eGFP-anillin expression cassette (AseI, HpaI) in a bluntended ligation. Lentiviral particles were prepared as described previously 32. The functionality of the lentivirus was tested by transduction of active murine fibroblasts and primary embryonic cardiomyocytes prepared from CD1 embryos at the embryonic stage E13.5 by collagenase-dissociation. The cells were incubated with the virus in DMEM supplemented with FCS (15% v/v), nonessential amino acids (0.1 mm), penicillin and streptomycin (50 µg/ml each) and β-mercaptoethanol (0.1 mm). After 24 h the medium was exchanged and 24 h later the cells were fixed for immunohistochemistry. Generation of transgenic mice Transgenic mice were generated by aggregation of transgenic G4 ESCs from two individual egfp-anillin clones with tetraploid CD-1 embryos. Two-cell stage embryos from superovulated CD-1 mice were flushed on day 2.5 post coitum. Embryos were electrofused in 0.3 M mannitol using a pulse generator CF-150/B (BLS), applying two 28V pulses for 45 µs each. Fused embryos were cultured overnight in KSOM medium (Millipore) at 37 C, 4% CO 2. Transgenic egfp-anillin ESC clones were trypsinized and aggregated with two 4-cell stage tetraploid embryos in KSOM medium after removing of their zona pelucida in Tyrode s solution (Sigma). Aggregated embryos were incubated overnight at 37 C, 4% CO 2 and transferred into uteri of pseudo- pregnant CD-1 foster mice on the following day. Transgenic mice were crossed with CD-1 mice for testing germline transmission. Offspring with agouti coat colour was analysed for inheritance of the transgene by PCR with the following primer pair: anillin-5 -long 5 -GGCACAAGCTGGAGTACAAC-3 and anillin-3 -long 5 -

2 TGGCACTGGTGCAAAGTATG-3. The two obtained transgenic egfp-anillin mouse lines did not display any obvious differences with respect to transgene expression pattern. For most of the experiments hemizygote mice were used, their egfp-anillin expression pattern was very similar to homozygotes. Determination of cell cycle kinetics Measurements of cell cycle durations were done on the time lapse sequences of of egfpanillin transgenic murine and human pluripotent cells, embryonic hearts and acute brain slices. Duration of karyokinesis was determined as the time interval between nuclear membrane dissolution and appearance of the contractile ring; duration of cytokinesis was determined as the time period in which a contractile ring was visible; M-phase duration was calculated by adding the durations of karyokinesis and cytokinesis; early G1-phase was defined as the time interval from the end of cytokinesis to the first reappearance of egfpanillin fluorescence in the nucleus; cell cycle duration was determined by monitoring dissolving of the nuclear membrane in the mother and daughter cells, respectively. Determination of DNA-content by confocal laser microscopy PFA-fixated, cryopreserved hearts from CAG-eGFP-anillin mice at 12 days after cryoinfarction were sliced into 50 µm thick sections with a cryotome CM 3050S (Leica) and treated with RNAse A (20 µg/ml) in wash buffer (0.5 M NaCl, 0.1 M Tris ph 7.5, 50 mm EDTA) for 1 h at 37 C. For DNA staining, slices were incubated with 1 µm TO-PRO 3 iodide (642/661) (Molecular Probes) at RT for 2 h. Cardiomyocytes were identified by crossstriation in the DIC channel; egfp-anillin + -cardiomyocytes could also be identified by their prominent nuclear egfp-fluorescence. Endothelial cells were identified by immunostaining for PECAM-1 (1:800, Becton Dickinson) and Rhodamine wheat germ agglutinine (1:100, Vector Laboratories). Image stacks were acquired with an inverted confocal laser scanning

3 microscope (Nikon Eclipse Ti) equipped with a x40/1.15 NA water dipping objective (ApoLWD 40x WI SDIC N2). Stacks of 18 to 48 images (512x512 pixels) with a z-step width of 0.5 µm were recorded. Excitation wavelength for egfp-anillin, Cy3/Rhodamine and TO- PRO 3 were 488 nm, 543 nm and 642 nm, respectively. For DNA content analysis NIS- Elements AR bit software was used. The sum of pixel intensities of the TO-PRO 3 signal within the manually defined nucleus area was measured in every single z-plane; the sum of these values corresponds to the DNA content 33. Cell culture of murine and human pluripotent cells G4 hybrid ESCs 34 were cultured in Knockout-Dulbecco s modified Eagle s medium (DMEM) (Invitrogen), high-glucose, supplemented with 15% v/v fetal calf serum (FCS) (Promega), 0.1 mm nonessential amino acids (Invitrogen), 2 mg/ml L-Glutamine (Invitrogen), 50 µg/ml each penicillin and streptomycin (Invitrogen), 0.1 mm β-mercaptoethanol (Sigma), and 500 U/ml Leucemia inhibitory factor (LIF) (Chemicon). The ESCs were kept on irradiated murine fibroblasts derived from neomycin resistant mice. For generation of transgenic ESCs 5 x 10 6 cells were mixed with 30 µg of linearized plasmid DNA in PBS and electroporated at 250 V and 500 µf, 1 pulse, using a Bio-Rad Gene Pulser. The cells were plated on two 100-mm plates. Selection for neomycin resistant cells started two days after electroporation by adding 165 µg/ml G418 to the medium. Resistant colonies were picked onto mouse embryonic fibroblast-coated 24-well plates, propagated and analyzed for egfp expression. ESC differentiation was performed using the Hanging Drop protocol. In brief, 0.2 x 10 6 ES cells were suspended in 10 ml Iscove s Modified Dulbecco s Medium (IMDM) (Invitrogen), supplemented with 20% FCS (Invitrogen) in the absence of LIF. 20 µl drops were incubated for 2 days at 37 C, 5% CO 2. The EBs were washed in bacterial dishes and cultivated with differentiation medium in suspension on a shaker. After 3 days EBs were plated separately on gelatine-coated glass cover slips in 24-well tissue plates or continuously cultivated in

4 suspension. At all stages of differentiation, EBs were monitored using a fluorescent microscope (Axiovert 200M, Carl Zeiss MicroImaging, Inc.) equipped with a FITC/TRITC filter set and 5 or 10 objectives. For isolation, at different time points of differentiation the EBs were washed and dissociated with 0.125% Trypsin/EDTA for 5 min. The treatment was stopped with differentiation medium containing FCS and the cells were centrifuged (210x g, 5 min). ~10 5 cells were plated on coverslips, pre-treated with 0.1% gelatine in 24-well tissue plates. After 48 h the cells were fixed with 4% paraformaldehyd (PFA) at RT for 30 min. Human ips cells were generated by retroviral transduction of human cord blood derived unrestricted somatic stem cells with OCT4, SOX2, KLF4 and cmyc and further cultivation 35,36. The human ips cells and H9 human ESCs 37 were transduced with CAGeGFP-anillin lentivirus on matrigel in conditioned media by single round infections for 24 h. Work with human ES cells (H9/WA09) was approved by RKI (Robert-Koch-Institut) license 37 from January 20th 2009 and performed at the MPI Münster. Culture and differentiation of TS-cells Rs26 TS-cells (a kindly gift of J. Rossant, Toronto) were transiently transfected using the Lipofectamine LTX and Plus reagent (Invitrogen) and cultured in RPMI 1640 (life Technologies) supplemented with 20% fetal bovine serum (PAN-Biotech), sodium pyruvate (1 mm) (Gibco), β-mercaptoethanol (100 µm) (Sigma), L-glutamine (2 mm) (Life Technologies), and penicillin and streptomycin (50 µg/ml each, Life Technologies). Human recombinant FGF4 (25 ng/ml) (Sigma) and heparin (1 µg/ml) were added to the TS cell medium directly before use 10. Differentiation to trophoblast giant cells was initiated by withdrawal of FGF4 and addition of CDK1-inhibitor RO3306 (final concentration 6 µm 11 ).

5 Supplementary References 32. Pfeifer, A., Ikawa, M., Dayn, Y. & Verma, I. M. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. U. S. A 99, (2002). 33. Ploeger, L. S. et al. Implementation of accurate and fast DNA cytometry by confocal microscopy in 3D. Cell Oncol. 27, (2005). 34. George, S. H. et al. Developmental and adult phenotyping directly from mutant embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A 104, (2007). 35. Kim, J. B. et al. Direct reprogramming of human neural stem cells by OCT4. Nature 461, (2009). 36. Zaehres, H. et al. Induction of pluripotency in human cord blood unrestricted somatic stem cells. Exp. Hematol. 38, , 818 (2010). 37. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, (1998).