ES cell culture
MC1 ES cells, derived from 129S6/SvEvTac47
, were purchased from the Transgenic Core Laboratory of the Johns Hopkins University School of Medicine, Baltimore, MD, USA. R26R317
ES cells, a gift from Dr. Philippe Soriano, were used as parental line to generate pZscan4-CreERT2 cells for lineage-tracing experiments. In general, all ES cell lines were cultured for 2 passages on gelatin-coated feeder-free plates and subsequently maintained in gelatin-coated 6-well plates in complete ES medium: DMEM (Gibco); 15% FBS (Atlanta Biologicals); 1000 U/ml leukemia inhibitory factor (LIF) (ESGRO, Chemicon); 1 mM sodium pyruvate; 0.1 mM non-essential amino acids (NEAA), 2 mM GlutaMAX, 0.1 mM beta-mercaptoethanol, and penicillin/streptomycin (50 U/50 µg/ml). For all cell lines, medium was changed daily and cells were split every 2 to 3 days routinely.
pZscan4-Emerald vector construction
A putative Zscan4 promoter that covers between the Zscan4c start codon (methionine) and the 2570 bp upstream site from the start codon was amplified from BAC RP23-63I1 with high fidelity TITANIUM Taq polymerase (Clontech) using primers (Forward primer, MluI_site_GGCAACCTTACTACTTCTATC; Reverse primer, AGCATCAACCACTTTGGTAC). Subsequently, the PCR product was cloned into the pCDNA6.2/C-EmGFP TOPO vector (Invitrogen). Sequence-verified plasmid DNA was linearized by MluI digestion.
pZscan4-CreERT2 vector construction
An open reading frame (ORF) of pCreERT216
vector was cloned into Eco
RI site of pBluescriptIISK(+) plasmid vector. Subsequently, the aforementioned PCR fragment of Zscan4c promoter was cloned into the vector between Eco
RV and Eco
RI sites, located at the 5’ end of the CreERT2-ORF by blunt-end ligation. A Sal
I DNA fragment containing Zscan4c promoter-CreERT2 was cut out from the vector and then cloned into pEF6/V5-His-Topo plasmid vector between Hind
III and Pme
I sites by blunt-end ligation.
Generation of pZscan4-Emerald and pZscan4-CreERT2 cells
MC1 ES cells (for pZscan4-Emerald transfection) and R26R3 ES cells (for pZscan4-CreERT2 transfection) were grown on gelatin in 6-well plates. 5×105 cells in suspension were transfected with 1 µg of linearized pZscan4-Emerald vector or pZscan4-CreERT2 vector using Effectene (QIAGEN) according to manufacturer’s instructions, and plated in gelatin-coated 100 mm dishes. Cells were selected with 5 µg/ml blasticidin. ES cell colonies were picked on the 8th day, expanded, and frozen for further analysis.
Sorting pZscan4-Emerald ES cells
Cells were fed at least 2 hours before harvesting by Accutase (Chemicon), and resuspended in IMDM containing 25 mM HEPES buffer (Chemicon) with 1% fetal bovine serum and 1000 U/ml LIF. The cells were then FACS-sorted according to the fluorescent intensity of Emerald into IMDM containing 35% serum, 1 mM sodium pyruvate, 2 mM GlutaMAX, 100 µM β-mercaptoethanol, 100 U/ml Penicillin, 100 µg/ml streptomycin, 0.1 mM non-essential amino acids, and 1000 U/ml LIF. For qRT-PCR analysis of FACS-sorted cells, total RNAs were collected immediately after sorting by TRIzol (Invitrogen) according to the manufacturer’s instructions.
LacZ analysis of pZscan4-CreERT2 cells
To trace the fate of Zscan4+ cells, pZscan4-CreERT2 ES cells were grown in the standard ES medium in the presence or absence of 100 nM tamoxifen for up to 9 passages. As a control, pZscan4-CreERT2 cells were also cultured in the presence of tamoxifen for 3 passages, followed by 6 additional passages without tamoxifen. To visualize the LacZ+ cells, cells in biological triplicate at passages 1– 9 were stained for beta-galactosidase (LacZ) using a commercial kit (Chemicon) according to manufacturer’s instructions. For flow cytometry analysis, LacZ was also detected by the green fluorescence substrate CMFDG using the commercial kit DetectaGene™ (Invitrogen). Suspended cells were then analyzed by the Guava EasyCyte Mini flow cytometry system with the CytoSoft4.1 software (Guava Technologies).
Embryoid body (EB) formation assay of pZscan4-CreERT2 cells
To examine the difference between Zscan4+ and Zscan4− cells for their ability to differentiate into multiple cell lineages, EB formation assay18
was carried out after culturing pZscan4-CreERT2 ES cells on gelatin for 3 days in the complete ES medium containing 100 nM tamoxifen. Cells were then harvested and 4×106
ES were plated on 100 mm bacteriological Ultra-Low Culture Dish (Corning) in LIF-free medium without tamoxifen to form floating EB. On the 7th day floating EB were collected and plated in gelatin-coated 6-well plates in LIF-free medium without tamoxifen to allow attachment. On the 11th
day, areas of beating muscles were scored and subsequently cells were fixed in 4% PFA for further analyses by LacZ staining and immunohistochemistry.
Mouse chimera assay of pZscan4-Emerald cells
Eight-week-old C57BL/6J mice were superovulated by injecting 5 IU pregnant mare serum gonadotropin (PMS; Sigma, St Louis, MO, USA) and 5 IU human chorionic gonadotropin (HCG; Sigma) after 46–47 h51
. Fertilized eggs were harvested from mated superovulated mice and cultured in synthetic oviductal medium enriched with potassium (KSOMaa MR-121-D) at 37°C in an atmosphere of 5% CO2
. Emerald-positive cells were collected by FACS-sorting pZscan4-Emerald cells and subsequently microinjected into 3.5 d.p.c. blastocysts. 15 blastocysts were transferred into the uteri of 2.5 d.p.c. pseudopregnant ICR female mice. Embryos were collected at day 10.5 d.p.c. and genotyped for chimerism by PCR.
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
1 µg of total RNAs, isolated from cells by TRIzol (Invitrogen) in biological triplicate, were reverse transcribed by Superscript III according to the manufacturer’s instructions. qPCR analyses were performed using 10 ng of cDNAs per well in triplicate with the SYBR green master mix (Applied Biosystems) according to the manufacturer’s instructions. Primers used: Zscan4 (recognize all Zscan4 paralogs; forward, 5’- GAGATTCATGGAGAGTCTGACTGATGAGTG-3’; reverse, 5’-GCTGTTGTTTCAAAAGCTTGATGACTTC-3’); Zscan4c (forward, 5’-CCGGAGAAAGCAGTGAGGTGGA-3’; reverse, 5’-CGAAAATGCTAACAGTTGAT-3’); Zscan4d (forward, 5’-GTCCTGACAGAGGCCTGCC-3’; reverse, 5’-GAGATGTCTGAAGAGGCAAT-3’); Spo11 (forward, 5’-GCTGGACAGCATCCTGAAGAGG-3’; reverse, 5’- GGGTAAGTACACTCTGGACA-3’); Dmc1 (forward, 5’-CAGATCCAGGAGCAACTATGA-3’; reverse, 5’-CGATCCTCAGTTCTCCTCTT-3’); and Smc1β (forward, 5’- GCATCCATCCAAATAGACTACAGC-3’; reverse, 5’-CCTGTGCTCGCAAGTTTGGA-3’). Reactions were run on 7900HT or 7500 system (Applied Biosystems). Fold induction was calculated and normalized by the ΔΔCt method52
Generation of tet-Zscan4c and tet-Empty cells
The detailed procedures for making tet-Zscan4c and tet-Empty ES cells were described previously19
. In brief, “MC1 Rosa26 knock-in parental ES cell clone” was made by targeting ROSA26 locus17
of MC1 ES cells47
by pMWRosaTcH vector48
(a gift from Dr. Hitoshi Niwa). The ORF of Zscan4c was amplified by PCR and subcloned into the pZhcSfi plasmid48
(a gift from Dr. Hitoshi Niwa) after replacing the Zeocin-resistance gene by puromycin-resistance gene19
. A 6xHis-FLAG epitope tag sequence was inserted into the 5’ end of LoxPV site to make ZSCAN4C fused to the epitope tag at the C-terminus19
. The resultant Zscan4c plasmid was cotransfected with pCAGGS-Cre plasmid into the MC1 Rosa26 knock-in parental ES cells using Effectene (QIAGEN) according to manufacturer’s instructions. After the puromycin selection in the presence of Doxycycline (0.2µg/ml), ES cell clones were isolated and thereafter named tet-Zscan4c cells. Modified pZhcSfi vector without ORF was used to establish tet-Empty control cells.
Telomerase knockdown in tet-Zscan4c cells
The tet-Zscan4c cells were transiently transfected with a SMART pool siRNA (Dharmacon) against mouse Tert gene (the catalytic protein unit of the enzyme telomerase). Non-targeting control (NTC) siRNA pool (Dharmacon) was used as a control for off-target effect. The effects of Zscan4 overexpression in these cells were examined by subsequent induction of Zscan4 in the Dox− condition for 3 days. Tert knockdown was confirmed by qRT-PCR analysis and TRAP assay.
Generation of tet-Zscan4c-KR cells
After testing four different shRNA designs against the sequence common to 3’-UTRs of both Zscan4c and Zscan4d, the following shRNA sequence (linker, 19-mer sense oligos, a hairpin loop, and an anti-sense of the 19-mer sense oligos, and an antisense of the linker; see Supplementary Fig. 6
) was selected and used: forward, 5’-CATAACCTGAAAAAACAGAAGCCTGGCATTCCCTAAGCTTAGGGAA TGCCAGGCTTCTGCGCGTCCTTTCCACAAGA TATATA-3’; reverse, 5’-CATAACCTGAAAAAACAGAAGCCTGGCATTCCCTTTCGAAAG GGAATGCC AGGCTTCTGCGCGTCCTTTCCACAAGATATATA-3’. The shRNA was cloned into GeneSilencer U6 PCR kit (Gelantis) according to the manufacturer’s instructions. The resultant vector was stably transfected into the tet-Zscan4c cells by the Effectene (QIAGEN) according to manufacturer’s instructions. The cells were named tet-Zscan4c-knockdown/rescue (KR) cells, as the endogenous Zscan4 was knocked down in the Dox+ condition and the exogenous Zscan4c provided a rescue in the Dox− condition. As a negative control, an shRNA against Luciferase provided by the kit was stably transfected into tet-Zscan4c cells and named shCont cells.
Telomere Quantitative Fluorescence In Situ Hybridization (Q-FISH)
All the cells were maintained in the complete ES medium containing Dox, except for Zscan4-overexpression condition achieved by culturing cells in the Dox− condition for 3 days. Medium was replaced every day. On the 3rd
day medium was supplemented with 0.1 µg/ml colcemid (Invitrogen), followed by 4 hours incubation to arrest the cells in metaphase. After adding hypotonic 0.075 M KCl buffer, cells were fixed in cold methanol/acetic acid (3:1) and metaphase spreads were prepared. Telomere FISH was performed by Telomere peptide nucleic acid (PNA) FISH Kit/Cy3 (DakoCytomation) according to the manufacturer’s instructions. Chromosomes were stained with 0.5 µg/ml DAPI. For quantitative measurement of telomere length, digital images of chromosomes and telomeres were captured by Zeiss microscope with Cy3–DAPI filter sets, followed by the quantitation of telomere size and fluorescence intensity by TFL-TELO software 22
Telomere Chromosome Orientation FISH (CO-FISH)
CO-FISH analysis was done as described 24,49
with several minor modifications. Briefly, ES cells were grown in either Dox+ or Dox− condition for 3 days. Medium was changed every day. On the 3rd day 5'-bromo-2'-deoxyuridine (BrdU) was added for 12 hours to allow BrdU incorporation for one cell cycle. Colcemid (0.1 µg/ml) was added for the final 4 h. Metaphase spreads were prepared. Slides were stained with 0.5 µg/ml Hoechst 33258 (Sigma), washed in 2× SSC for 20 min at room temperature, mounted with McIlvaine’s buffer (at pH 8.0), and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 min. The BrdU-substituted DNA was digested with 3 units/µl Exonuclease III (Promega) for 10 min at room temperature. The leading strand telomeres were revealed by 3'-Cy3-conjugated (TTAGGG)7
(IDA) without denaturation step and incubated overnight at 37°C. Chromosomes were counterstained with 1 µg/ml DAPI (Vector Laboratories).
Telomere measurement by quantitative real-time PCR
Genomic DNA was extracted from 106
cells and quantified by Nanodrop. Average telomere length ratio was measured from total genomic DNA using a real-time PCR assay, as previously described 21
. PCR reactions were performed on the Prism 7500 Sequence Detection System (Applied Biosystems) using telomeric primers, control single-copy gene Rplp0, and PCR settings as previously described 21
. A standard curve was made for reference gene by serial dilutions of known amounts of DNA from 100 ng to 3.125 ng. The telomere signal was normalized to Rplp0 to generate a T/S ratio indicative of relative telomere length.
Immunohistochemistry with Telomere FISH
High-quality metaphase spreads were prepared as described for Q-FISH method. Slides were unmasked for 1 hour 30 min at 90°C, dehydrated, and incubated for 5 min at 83°C with Telomere Probe Alexa488. Slides were allowed to anneal at room temperature for 1 hour. Slides were incubated at 4° C overnight with primary antibody in block solution. Primary antibodies used: Rabbit anti Zscan4 (1:5000), Mouse anti H2AX (1:1000), Goat anti SPO11 (1:400), Goat anti-DMC1 (1:200). Slides were incubated for 1 hour at room temperature with secondary antibodies (diluted in block solution): Alexa 568 Donkey anti Rabbit (1:800), Alexa647 Donkey anti-Mouse (1:200), Alexa569 anti-Goat (1:800). Cells were visualized by Zeiss 510-confocal microscope after staining nuclei with DAPI for 10 min at room temperature.
Telomerase activity measurement
All the cells were cultured in triplicate on gelatin-coated dishes for 3 days in complete ES medium in the presence (Dox+) or absence of doxycycline (Dox−). Cell lysates were prepared from 106 cells per sample. Telomerase activity was measured by TRAP assay using a TRAPEZE Telomerase Detection Kit (Millipore) according to the manufacturer’s instructions.
G-banding karyotype Analysis
ES cells were treated with 0.1 µg/ml colcemid (Invitrogen) for 2 hour to induce metaphase arrest. Metaphase chromosome spreads were prepared, slides were incubated overnight at 65°C, treated with 0.1% trypsin for 1 min, and stained with 3% Giemsa reagent for 15 min. Karyotype analysis was carried out by SmartType software for n≥65 metaphases per sample (see ).
Sister Chromatid Exchange (SCE) assay
SCE assay was done as previously described in details 50
. Briefly, all mouse ES cells were maintained in complete ES medium containing doxycycline. For Zscan4 induction, doxycycline was removed from the medium for total of 3 days. Medium was added with BrdU for the last 24 hours, allowing the cells to complete 2 cell cycles. Medium was supplemented with 0.1 µg/ml colcemid for the last 4 hours to arrest the cells in metaphase. Metaphase spreads were prepared and SCE were counted in n>50 metaphases per sample. 3–4 independent experiments were carried out for each sample (total of n>150 metaphases).
Cells were plated in 24-well plates on sterilized coverslips. Cells in duplicate were maintained in Dox+ condition, whereas cells in triplicate were maintained in Dox− condition for 3 days to induce Zscan4 overexpression. Medium was changed every day. Cells were either fixed in 4% PFA for 10 min at room temperature or taken for metaphase spreads as described above. Cells in PFA were permeabilized with 0.25% NP-40 for 10 min. Cells were blocked for 10 min at room temperature in 1% BSA, 10% fetal bovine serum, and 0.2% saponin and incubated overnight at 4°C with the primary antibodies in a blocking solution: anti-FLAG antibody (1:1000), anti-ZSCAN4 (1:1000), anti-SPO11 (1:200), anti DMC1 (1:200), anti-TRF1 (1:500), anti-TRF2 (1:400), γ-H2AX (1:1000). As negative controls, cells stained without primary antibody were used as well as the Dox+ cells stained with Anti-Flag antibody. The bound antibody was visualized with a fluorescent Alexa546 secondary antibody (Invitrogen) under a Zeiss 510-confocal microscope. Nuclei were visualized with DAPI (Roche) staining for 5 min at room temperature.
Generation of Zscan4 antibodies
Custom-made polyclonal Rabbit anti-Zscan4 antibodies were generated (Genscript) against the C-terminal epitope of Zscan4: CSTYHRHLRNYHRSD (the C-terminal cysteine was added for KLH conjugation). This peptide is specific and common for predicted amino acids sequence of all Zscan4 paralogs, and thus, the antibody can recognize all the ZSCAN4 paralogs.