PCR for all cloning was performed with Q5 polymerase (NEB) according to manufacturer’s protocol. Plasmids involving lentiviral components were transformed into Stbl3 E. coli, other plasmids into DH5a. All enzymes used were from NEB and all cloning primers and oligos are listed in Additional file 2: Table S1.
Full length SHOX promoters were amplified from 293 T genomic DNA using Q5 polymerase and cloned to pGL3-Basic luciferase plasmid (Promega) using SacI;HindIII and MluI;HindIII sites for promoter 1 and 2, respectively. WRN, WRN mutants and BLM expression plasmids (pCMV-WRN/BLM) were obtained as a gift from Dr. Vilhelm Bohr, NIA. Vector control (pCMV-vector) was generated by replacing WRN with linker using NotI;XhoI sites. Mutagenesis on pGL3 containing SHOX P2 promoter was performed using Q5 Site Directed Mutagenesis Kit (NEB) according to manufacturer’s protocol. Full length SHOX was amplified from 293T cDNA and cloned into pHAGE-EF1a-IRES-zsGreen (Addgene #114008) using NotI;BamHI sites, creating pSHOX-GFP.
Mutant MSC and 293T cells were generated using 2-part lentiviral pCas9-Puro and pgRNA-GFP/Cherry system. pCas9-Puro were generated by Gibson assembly using PCR products of primers CP-1 on PX458 (Addgene #48138) and CP-2 on pLenti-TRE3G-BE3RA-PGK-Puro (Addgene #110846) and backbone of ClaI;NotI cut pHAGE-EF1a-IRES-zsGreen. pgRNA-GFP was constructed by inserting a gRNA cassette cloned from pDD-Cas9 (Addgene #90085) into pHAGE-EF1a-IRES-zsGreen with SpeI site. gRNA was inserted by golden gate cloning using BsmBI. All gRNA used in this paper are listed in Additional file 2: Table S2. Lentiviral WRN and WRN mutant overexpression carrying mCherry (pWRN-Cherry) were generated from Gibson assembly of 5 fragments specified in Additional file 2: Table S1 including a backbone fragment cut from NotI;MluI of pLX209-WRN (Addgene #125788). Vector control (pVector-Cherry) was generated by PCR of pWRN-Cherry without WRN sequence and ligating with NdeI site. Cherry version of pgRNA and pSHOX was generated by replacing GFP with mCherry cloned from phSyn-mCherry (Addgene #114472) into NdeI;ClaI cut backbone.
Human ESC H1 were maintained in mTESR+ (Stemcell) with normicin on geltrex (Gibco) coating with medium change every 2 days. H1 was passaged by dispase upon 70–80% confluency. MSC were cultured in DMEM (low glucose) medium supplemented with 1 × L-glutamine, 1 × NEAA, 10% MSC-qualified FBS and 1 × PSA (all from Gibco), with medium change every 2 days. MSC was passaged by Tryple Express upon 90% confluent. 293T cells were maintained in DMEM (high glucose) medium supplemented with 1 × L-glutamine, 1 × NEAA, 1 × sodium pyruvate, 10% FBS and 1 × PSA (all from Gibco).
Generation of mutant hESC and chondrogenic differentiation
hESC mutant lines were generated by nucleofection of H1 cells with Cas9/CRISPR PX458 plasmids (Addgene #48138) targeting WRN and SHOX, respectively using Human Stem Cell Nucleofector Kit 1 (Lonza) with A023 program. After nucleofection, GFP-positive cells were FACS sorted and replated in geltrex-coated plates in the presence of ROCK inhibitor Y27632 (Stemcell). Cells were allowed to grow into colonies, expanded and genotyped. The sequences of gRNA used were shown in Additional file 2: Table S2. Chondrogenic differentiation was performed according to published protocol . Since H1 was maintained in feeder-free condition, they were trypsinized to 2.5 × 104/cm2 on geltrex coating supplemented with ROCK inhibitor one day prior to differentiation. Other minor modifications included no passage on day12 and extension of differentiation to 18 days to promote chondrogenicity.
Wild-type H1 hESCs and WRNKO-hESCs were first dissociated by Accutase and seeded at a density of 1 × 104 cells/cm2 in osteogenic induction medium for 21 days. Osteogenic induction medium was changed every 2 days and was comprised of 90% knockout DMEM (Gibco), 10% heat-inactivated FBS (Gibco), 1% penicillin-streptomycin (Gibco), 1% GlutaMAX supplement (Gibco), 0.1 μM dexamethasone (Sigma), 100 mM β-glycerophophate (Sigma) and 50 μM ascorbic acid (Sigma). Expression of osteoblast differentiation was determined by alkaline phosphatase, Alizarin Red S staining and qPCR assay on day 7, 14 and 21, respectively.
293T was passaged and seeded in 15 cm dish (14 million cells each). In the next day, second generation lentivirus packaging plasmids were transfected in molar ratio of (Transfer: pMD2G: psPAX2 = 1.5: 1: 1) of 78 µg total DNA with 117 µL of Lipo8000 (Beyotime) in 6.5 ml OPTI-MEM. Lentiviral medium was collected 48 and 72 h post-transfection, filtered with 0.45 µM membrane and addition of PEG8000 (Santa Cruz) to 5% final concentration. After overnight incubation at 4 °C, virus was centrifuged at 1500g × 30 min and the pellet was resuspended in PBS to 50–100X concentration.
Gene rescue in MSC by lentiviral transduction
MSC was trypsinized with TrypLE, 0.5 × 106 MSC was incubated with concentrated lentivirus in 8 ug/ml polybrene in 5 ml medium per 10 cm dish overnight. Medium was changed the next day. Control and SHOX−/− MSC was generated by first transduced with Vector-Cherry lentivirus. One week later, they were transduced with Cas9-Puro and gRNA-GFP (scramble or SHOX gRNA) lentiviruses. MSC was FACS sorted for gRNA-GFP and puromycin (1 µg /ml) selected for Cas9 for 6 days.
For G4 rescue MSC, they were first transduced with Cas9-Puro lentivirus and selected as above. gRNA-GFP (G4 targeting or scramble gRNA) as well as gRNA-RFP (WRNKO or scramble gRNA) lentiviruses were transduced and FACS sorted for GFP/RFP + cells.
MSC chondrogenic differentiation
MSC differentiation was performed according to published protocol . Differentiation was initiated in MSC cultured for no more than 17 days post-lentiviral transduction.
G4 slot blot
Single-stranded DNA (ssDNA) oligos of 50 bp long were purchased commercially. Oligos were diluted to 15 uM in 100 mM KCl to final 200 µL. Diluted oligo of 100 µL per tube was heated at 95 °C for 10 min and gradually cooled to 20 °C at 0.1 °C/sec using PCR machine (C1000 Touch, Bio-Rad). Nitrocellulose membrane (0.45 um, Thermo) and filter papers were pre-wet for 10 min in TBS. After assembly of Slot blot apparatus (1706542, Bio-Rad), 200 µL TBS were applied to each well and vacuumed through. 200 µL of samples or TBS were applied to each well and allowed to slowly pass through membrane on low power vacuum, taking about 10 min to completely drain. Membrane was then briefly dried and baked at 80 °C for 2 h for DNA immobilization. Membrane was then blocked with 10% horse serum, 3% BSA in TBS for 1 h, followed by incubations with BG4 antibody (1:1000) for 1.5 h at RT, anti-FLAG antibody (1:1000) overnight at 4 °C and anti-mouse HRP (1:2500) for 1 h at RT. Membrane was washed 3 times after each antibody incubation with 0.5% Tween/PBS. Antibodies were diluted in 0.5% Tween/PBS. ECL substrate (Bio-Rad) were applied to membrane and exposed with ChemiDoc (Bio-Rad).
293T cells were passaged 1 × 105 per well in 48-well plate. In the following day, medium was changed and 0.6 µg of overexpression plasmids (pCMV-WRN/BLM, a gift from Dr. Vilhelm Bohr, NIA) were transfected using 0.8 µL Lipo8000 (Beyotime) in each well. After overnight incubation, medium was changed and transfected with 0.4 µg pGL3-basic luciferase plasmid (Promega) containing SHOX promoter sequences, 0.04 µg pRL-TK renilla plasmid and 0.6 µL Lipo8000 per well. Next day, cells were digested using Dual-Glo Luciferase Assay System (Promega) according to manufacturer’s protocol. Luciferase and renilla reading were taken by SpectraMax i3x (Molecular Devices) plate reader.
293 T mutant generation and ChIP-qPCR
293T cells for ChIP assay was transduced with Cas9-Puro and gRNA-GFP (WRNeij gRNA) lentiviruses then FACS sorted, same method as MSC transduction. These WRNKO 293T were then seeded on 10 cm dish and transfected with pCMV-FLAG-WRNK577M using Lipo8000 (Beyotime). Cells were harvested 2 days post-transfection for ChIP. ChIP protocol was performed according to published protocol . Briefly, 1 × 107 293T cells were fixed with 1% PFA on dish for 10 min at RT. Then cells were quenched with glycine and chromatin extracted using Chromatrap Sonication Shearing Kit (Chromatrap) according to manufacturer’s protocol. Chromatin was sonicated with SLPe sonicator (Branson) with 2.4 mm tip diameter in 60 V (on:10 s, off:60 s) for 11 min on ice. Sonicated chromatin was diluted tenfold, 30 µL was stored as input while 900 µL were immunoprecipitated with 50 µL FLAG-beads (Sigma) or IGG-beads (CST). After overnight incubation at 4 °C, bound DNA was washed and eluted. Input and precipitated DNA were purified by phenol/chloroform and ethanol precipitation method. qPCR details were shown in Reverse transcription-qPCR section.
Cells were flash frozen and later lysed in Trizol (Invitrogen) according to manufacturer’s protocol. MSC-derived chondrogenic pellets were mechanically disrupted with pestles in 1.5 ml tube containing Trizol. RNA was collected by standard isopropanol precipitation. Reverse transcription was performed with Primescript RT master mix (Takara) according to manufacturer’s protocol. Quantitative PCR was performed using Powertrack SYBR MM (Invitrogen) in ABI QuantStudio 7 Real-time PCR System. Fast programme for cDNA samples and standard programme for CHIP DNA samples. Technical triplicate was performed on each sample. All primers for qPCR were listed in Additional file 2: Table S3.
Cells were lysed in RIPA lysis buffer (Beyotime) for 20 min at 4 °C. After centrifuge, supernatant of 20 µg total protein was diluted with 4X Laemmli buffer (Bio-Rad) and denatured at 100 °C for 10 min. Semi-dry transfer was performed with Trans Blot Turbo (Bio-Rad) using standard programme to 0.22 µM membrane. Membrane was blocked with 5% goat serum for 1 h at RT then in respective primary antibody overnight at 4 °C. In the following day, secondary HRP antibody was incubated for 1 h at RT. Washes (3 times) after each antibody incubation step were performed with 0.1% Tween in PBS. All antibodies were diluted in 5% goat serum and concentrations were listed in Additional file 2: Table S4. ECL substrate (Bio-Rad) were applied to membrane and exposed with ChemiDoc (Bio-Rad).
Tissue processing, immunofluorescence and histological staining
MSC derived chondrogenic pellets and cells were fixed in 4% PFA overnight and 10 min, respectively. Fixed pellets were immersed in 30% sucrose overnight, embedded in OCT and sectioned at 8 µm thickness with cryostat CM3050 (Leica). Pellet sections were pre-treated with 1 mg/ml Pronase E (Sigma) in 37 °C for 5 min prior to staining. Cells and sections were treated with 0.5% Triton × for 10 min prior to blocking with 10% goat serum for 1 h. Antibodies were diluted in 5% goat serum with 0.1% Tween-20 according to Additional file 2: Table S4. Primary antibody was incubated O/N at 4 °C and secondary antibody for 1 h at RT. DAPI (1 mg/ml) was incubated for 10 min RT. Prolong glass mountant was used according to manufacturer’s protocol. Fluorescence was imaged by either Ti-2E fluorescence microscope (Nikon) or FV1200 confocal microscope (Olympus). Same staining and imaging criteria were applied between mutants or groups. Images were adjusted by the same LUTs or brightness/contrast among comparing groups.
For histological stain, pellet section or fixed cells were wash with PBS and water, twice each. Cells were then incubated in 0.1% safranin-O solution (Milipore) for 10 min and washed with water for 5 times. Sections were incubated in 1% Alcian blue solution (Milipore) for 20 min RT. Sections were rinsed gently under tap water for 2 min, air-dried and dipped in xylene before mounting in resin mounting medium (Thermo). Histological staining was imaged by IX83 inverted microscope (Olympus).
DMMB GAG quantitative assay
GAG quantification was performed with DMMB dye according to published protocol . Briefly, samples were digested in 0.15 µg/µL Proteinase K (Thermo) in ammonium acetate buffer (PKAA) at 60 °C for 3 h. Shark chondroitin sulfate (Sigma) (0–1.5 µg) and calf thymus DNA (Sigma) (0–800 ng) were diluted in PKAA as standards. Samples were diluted 5- or 20-fold depending on GAG and DNA content. 200 µL DMMB (sigma) solution (0.016 mg/ml, pH 3) was added to each 20 µL sample, and OD525 measured immediately. 100 µL Hoechst 33258 (Sigma) (0.7 µg/ml) was added to every 100 µL samples and standards. Fluorescence measured with excitation: 340 nm; emission: 465 nm. All readouts were in taken with SpectraMax i3x (Molecular Devices) plate reader and with technical triplicate.
Statistics and BG4 foci counting
All statistics were calculated with MS Excel using 1 or 2-tailed Student’s t-test assuming unequal variance. Z-stacks of BG4 nuclei staining was maximum projected, brightness adjusted and set an equal threshold to all images. BG4 signals per DAPI were determined by ImageJ “Analyse particle” function with size of “0.01-infinity” with no circularity limitation.
Injection of Morpholino into zebrafish embryo and whole-mount staining
The use of zebrafish and animal experiments were approved by Animal Experimentation Ethics Committee of CUHK. Wild-type zebrafish (AB line) were maintained at 28.5 °C on a 14 h: 10 h light-dark cycle. Morpholinos (Gene Tools) were injected into the yolks of zebrafish embryos (10 ng/embryo) at one-cell stage using a gas-driven microinjector. The efficiency of Morpholino was visualized by the lissamine conjugated in Morpholino and the expressions of shox and wrn transcripts were measured by RT-qPCR and phenotypic penetrance. The injected embryos were cultured in E3 embryo media until 7dpf. Body length of each morphant was measured daily from 3 dpf. Morpholinos used in this study are: shox MO 5′-AGCGTGCAGAAGAAACTCACCGTCA -3′, wrn MO 5′-TTCCTGATGTCTGTGAAAACATATA-3′, and control MO 5′-CCTCTTACCTCAGTTACAATTTTATA-3′. For whole-mount staining, zebrafish embryos at 7dpf were collected and fixed in 10% formalin overnight. Cartilage was stained with Alcian blue solution. The morphants and controls were observed and imaged under Nikon SMZ800 stereo microscope and body length were measured by NIS element.