Generation of the knock-in mouse
We targeted the MBS cassette to the 3′UTR of the mouse β-actin gene, downstream of the zipcode. We identified a suitable region containing appropriate restriction sites for integration of the 24 × MBS cassette. We used a ~ 9.6 kb genomic region to construct the targeting vector that was first sub-cloned from a positively identified BAC clone containing the mouse β-actin gene. We designed the region such that the long homology arm (LA) includes the full β-actin gene, extending from ~ 4 kbp upstream of the β-actin transcription start site to ~ 750 bp downstream of the end of the last exon. The short homology arm (SA) extends 1.3 kb 3′ downstream of the Neo cassette (). The 1.3 kbp long MBS cassette is inserted 441 bps downstream to the stop codon.
We digested the MBS cassette on both ends using MluI and ligated the product into the MluI site of a vector containing the loxP flanked Neo cassette. We synthesized a PCR fragment from the genomic DNA using primers containing respectively MluI and AscI sites to generate the sequence between the site of the MBS cassette insertion and the site of the Neo cassette insertion. We ligated it into the vector between the MBS and the Neo cassettes. We excised the region including the MBS repeats, the intervening genomic sequence and the Neo cassette using BsiWI and ligated into the targeting construct. The exact genomic sequence is restored except for the insertion of the MS2 repeats and the floxed Neo cassette at the locations described above (). We confirmed the targeting vector by restriction analysis and sequencing.
The generation of the targeting vector, the ES cells and the heterozygous mice was performed by Ingenious Targeting Laboratory Inc. The targeting vector was linearized and transfected by electroporation into 129SVev ES cells. After selection in G418, we expanded surviving clones for PCR analysis in order to identify recombinant ES clones. We obtained three stable ES clones and verified the expression of the β-actin-MBS by RNA FISH and by co-expression of MCP-GFP in both undifferentiated and differentiated state. We injected positive ES cells into foster mothers and obtained chimeric litter (based on coat color). Two males showing 90% chimerism were mated with female mice, and 7 positive F1 germline mice (4 males, 3 females) were obtained from clone 3C-C3. We verified F1 mice by genotyping and PCR of the MS2 sequences, as well as RNA FISH in primary mice cells derived from mouse tissues. Experiments on mice were approved by the Institutional Animal Care and Use Committee of Albert Einstein College of Medicine (Protocol Number 20100908).
We created a NLS-MCP-YFP gene using PCR. The final construct adds an NLS and an HA tag to the N-terminus of the previously reported construct MCP-YFP31
. We cloned this expression cassette into the pHAGE-Ubc-RIG lentiviral vector32
from which the DsRed-IRES-GFP fragment had been excised using NotI and ClaI. We used this plasmid to create recombinant lentiviral particles generating expression of NLS-MCP-YFP driven from the human Ubiquitin C promoter in target cells.
Isolation of primary hippocampal neurons from mouse embryos
We isolated embryonic day E18 mice from the pregnant female. We excised the hippocampus from the brain and placed it into sterile cold Hank’s Balanced Salt Solution (HBSS, without Mg2+, without Ca2+ supplemented with 5 mM HEPES). We then dissociated tissue using a scalpel, resuspended it in 15 ml ice cold HBSS, and spun down (100 g for 1 minute). We resuspended the supernatant into 2 ml per brain HBSS supplemented with 1:10 of 2.5% Trypsin solution (Invitrogen), and incubated 20 minutes at 30°C. We added 1/9 volume of OMI (Ovomucid Trypsin inhibitor, 10 mg.ml−1 in PBS) and 1/19 volume of DNAseI (100 mg of DN25-100mg, Sigma resuspended in HBSS without Mg2+ or Ca2+ then sterile filtered) and the tube was left to incubate 5 min at room temperature. We resuspended the remaining tissue in DMEM + 10% FBS, further dissociated using a fire polished Pasteur pipette and allowed to settle 3 min (operation repeated up to 8 times). We counted and deposited cells on Ploy-Lysine coated coverslips, allowed them to attach (1 h at 37° C, 5% CO2), and replaced the culture medium by fresh Neurobasal medium (Invitrogen) supplemented with 1 × B27 (Gibco) 2 mM glutamax (Invitrogen), Primocin (Invivogen) and 25μM glutamate. We then half-changed the medium every 3–4 days (omitting glutamate), supplementing with 10μM Cytosine Arabinoside. To express NLS-HA-MCP-YFP in primary neurons, we applied lentiviral particles generated using the pHAGE vector to primary neurons by addition to the culture media 1 day after plating. We maintained cells under standard neuron culture conditions, and could see expression as early as 36 hours post infection.
Isolation of Mouse Embryonic Fibroblast Cell lines from mice
For the MEF cell lines, we isolated embryonic day E14 mice from the pregnant female. We separated the head from the body and used it to genotype each embryo. We removed the dark cardiac tissue from the body and digested the body with Trypsin EDTA for 20 min. We plated MEFs isolated from each embryo separately into a 10 cm dish and grew them for 1 day in DMEM + 10% FBS with antibiotics. We detached the cells with Trypsin and seeded them into a culture dish for immortalization or frozen in 10% DMSO in DMEM with 10% FBS. To express NLS-MCP-GFP in primary MEFs, we seeded cells on the day of their isolation on a fibronectin coated Labtek chamber (Thermo Scientific), and incubated them with lentiviral particles generated using the pHAGE vector.
To immortalize MEFs, we transfected the cells with a plasmid expressing SV40 Large T Antigen (pBSSVD2005, gift of David Ron) using Fugene 6 (Roche). We followed H.P. Harding protocol: after transfection cells were grown to confluence and then serially passaged at high and low densities at least 5 times in order to select transformed cells. To stably express MCP-YFP or MCP-GFP, we created recombinant lentiviral particles using the phage UbC plasmid (described above) and used them to infect these cells according to existing protocols. We used infected cultures after several passages to create a highly enriched population of stably expressing cells by FACS sorting of MCP-YFP fluorescent cells. We later selected individual clones from the MCP-YFP expressing cell lines.
We scraped MEF cells grown in DMEM 10% FBS 1% pen-strep from their dish. For tissue, we snap froze tissue in liquid nitrogen and grinded it using a pestle and mortar. We then extracted total RNA using the RNeasy mini kit (Qiagen). We diluted 20 μg RNA in 50% formamide, denatured it 5 min at 65° C, chilled it on ice and then loaded it on a non-denaturing 1% agarose gel in 0.5 × TBE. The RNA was transferred overnight onto an immobilon N+ membrane (Millipore). After UV-crosslinking, we incubated the membrane 1 h at 37° C in pre-hyb solution (6 × SSC, 50 mM NaPO4 pH 7.0, 5 × Denhardt solution, 4% SDS). We generated random primed gamma-32P-ATP (Perkin Elmer) labeled DNA probes using Klenow enzyme (Roche). We prepared the DNA templates by PCR of MEF cDNA for the wild type Actb and GAPDH probes, and used a cleaved-out MBS insert for the MBS probe. We diluted the DNA probe in 10 ml pre-hyb solution and incubated it with the membrane for 3 h at 37° C. We washed the membrane twice 20 min at 37° C in 7 × SSC − 50 mM NaPO4 pH 7.0 - 1% SDS and exposed it overnight to a storage phosphor screen, before imaging on a Storm 860 phosphorimager (Molecular Dynamics).
We performed RNA FISH on cultured cells to a protocol modified from 20, 33
. The probes used were 50mers of ssDNA bearing each 4–5 fluorophores (probe list in Supplementary Methods
). We grew MEF cells on coverslips in DMEM 10% FBS 1% pen-strep, then fixed them in 4% paraformaldehyde for 15 min at room temperature, before washing and storing in PBSM (PBS supplemented with 5 mM MgCl2) at 4° C. Prior to hybridization, we permeabilized the cells 10 min in 0.5% triton X100 in PBS, then washed them in PBS 10 min, and incubated 10 min in pre-hyb solution (50% formamide − 2 x SSC − 2 mg.ml−1
BSA - 0.2 mg.ml−1
tRNA - 0.2 mg.ml−1
sheared salmon sperm DNA). We then hybridized the probes to the cells for 3 h in pre-hyb solution supplemented with 10 ng DNA probe per locus per coverslip. We washed coverslips twice 20 min at 37° C with pre-hyb solution, then 10 min at room temperature in 2 × SSC, and 10 min at room temperature in PBSM. We counterstained DNA with DAPI (0.5 mg.l−1
in PBS). After a final wash in PBS, we mounted coverslips mounted on slides using ProLong gold reagent (Invitrogen). When 20mer probes were used, we replaced the pre-hyb solution by 10% formamide − 2 × SSC − 2 mg.ml−1
BSA - 0.2 mg.ml−1
tRNA - 0.2 mg.ml−1
sheared salmon sperm DNA − 10% dextran sulfate.
We performed RNA FISH in tissue section following a published method24
. Immediately after extraction from euthanized mice, we fixed the tissues in formalin overnight and paraffin embedded them. We used 5 μm thick sections for FISH. We incubated the sections at 55° C for 30 min, then submitted them to a high pressure treatment (30 s at 125° C, then 10 s at 90° C under ~ 18 PSI) in decloaker reveal reagent (Biocare medical). After 5 min incubation in H2
O, we briefly rinsed the slides in H2
O and PBS. We incubated the sections 20 min at room temperature in 0.25% NH4OH + 70% EtOH, 50 min at 4° C in freshly prepared 0.5% NaBH4
in PBS, then rinsed them in water then PBS. We incubated the slides 10 min at room temperature in PBSM, then 3 times 5 minutes in PBS, and finally 30 minutes in pre-hyb solution. After a 2 hour hybridization at 37° C in a closed chamber, we washed the slides 20 min at room temperature in pre-warmed (37° C) pre-hyb solution, 20 min at room temperature in pre-warmed (37° C) 2 × SSC, 20 min at room temperature in pre-warmed (37° C) 1 × SSC, 15 min at room temperature in pre-warmed (37° C) 0.5 × SSC, and 5 min at room temperature in PBSM (all washes performed on a slow rotary shaker). We counterstained the slides with 0.5 mg.l−1
DAPI, washed them once in PBSM before mounting on coverslips using Prolong gold reagent (Invitrogen).
We used as primary antibodies specific antibodies against β- and γ-actin isoforms (respectively 4C2F9H12/IgG1 and 2A3G8E2/IgG2b), a gift from Christine Chaponnier27
. We used as secondary antibodies goat anti-mouse IgG1 – FITC (Southern Biotech 1070-02), and anti-mouse IgG2b – TRITC (Southern Biotech 1090-03). We used a custom protocol27
: we incubated cells in prewarmed L-15 supplemented with 10% FBS, then fixed 20 min at room temperature in 1% paraformaldehyde in L-15, 10% FBS. We washed cells twice in PBS, permeabilized the cells with methanol (− 20° C, 5 min), and rinsed in PBS. We then incubated the cells 1 h in the primary antibody mix (PBS supplemented with 3% 0.2μm-filtered BSA fraction V, antibodies diluted 1:100 4C2F9H12/IgG1; 1:200 2A3G8E2/IgG2b) at 37° C in a closed chamber. We performed 5 rinses in PBS and incubated 1 h in the secondary antibody mix (PBS supplemented with 3% 0.2μm filtered BSA fraction V, 1:50 Southern Biotech 1070-02, 1:50 Southern Biotech 1090-03). After 5 rinses in PBS, we counterstained the DNA with 0.5 mg.l−1
DAPI and mounted on slides using Prolong gold reagent (Invitrogen).
We used the following primary antibodies: β-actin (Sigma A1978), γ-actin (AB3265, Chemicon) and β-tubulin (Amersham N357), and secondary antibodies: Donkey anti-mouse 800 nm (Rockland 610-732-124) and Donkey anti-sheep 700 nm (Invitrogen A21102). We washed cells in ice-cold PBS and lysed them at room temperature 2 min in 1 ml lysis buffer per 10 cm dish (50 mM Tris-Hcl pH 8.0, 150 mM NaCl, 1% NP40, 5 mM DTT, 1 mM PMSF, half a mini tablet protease inhibitor). We spun the lysate 15 min at 14000 g at 4° C and loaded the supernatant on a Nupage 4–12% bis-tris gel using MOPS running buffer (Invitrogen). After transfer on a nitrocellulose membrane in Nupage transfer buffer (25 V for 1.5 h), we blocked non specific interactions by incubating the blot overnight at 4° C in PBS supplemented with 5% non-fat dry milk. After that, we rinsed the membrane and incubated it 1 h with the primary antibodies in PBS supplemented with 1% BSA (dilutions: 1:2,500 mouse anti-β-actin; 1:5,000 mouse anti-β-tubulin). We then washed the blot 5 times 10 min in PBS + 0.3% Tween-20, before incubation for 30 min with the secondary antibody (1:10,000 in PBS + 1% BSA). We then washed the membrane 5 times in PBS + 0.3% Tween-20, before exposure on an Odyssey infrared imaging system (2 color detection). We quantified the bands intensities using ImageJ software (NIH).
For Immortalized cells, we plated homozygous MEFs expressing MCP-YFP on a 0.17 mm delta T dish (Bioptechs) and incubated 24 h in DMEM 10% FBS 1% pen-strep. We left cells to recover for 24 h and then starved them overnight. We replaced the media by L-15 supplemented with the Oxyfluor oxygen scavenging system (Oxyrase) prior to the experiment. We placed the cells on an Olympus IX-71 microscope equipped with a 150 × 1.45 NA objective (Olympus) and a Cascade II camera (Photometrics). We maintained a constant temperature of 37° C through the experiment using the Delta T chambers, a heated lid and objective heater (Bioptechs). We imaged cells in three dimensions over time using a 200 nm z-axis step size over a range of 4 μm every 4 min using a 488 nm argon laser for excitation of YFP fluorescence. We converted the three dimensional z-stacks into two dimensional movies using a maximum intensity projection. For primary fibroblasts, we plated the cells on fibronectin-coated MatTek dishes (MatTek), infected with lentivirus to express NLS-MCP-GFP, and incubated them for 48 h in DMEM 10% FBS, 1% pen-strep. We stained the cells with 5 μM CellTracker Orange CMRA (Invitrogen) and replaced the media with L-15 containing 10% FBS, 1% pen-strep, and 1% oxyrase prior to the experiment. We used an Olympus IX-71 inverted microscope equipped with a 40 × 1.35 NA oil-immersion objective (Olympus), and EMCCD camera (Andor). We maintained the temperature at 37° C using an environmental chamber (Precision Plastics). We excited GFP using a 488 nm line from an argon ion laser (Melles Griot) and CellTracker Orange (Invitrogen) using a 561 nm diode laser (Cobolt). For RNA FISH, we imaged the slides on an Olympus BX-61 microscope equipped with an X-cite 120 PC Mercury lamp (EXFO), a 100 × 1.4 NA objective (Olympus) and a Coolsnap HQ camera (Photometrics). For primary neurons, we changed neuron media into Hibernate Low Fluorescence (BrainBits LLC) with 1 × B27 supplement and 2 mM Glutamax. Post imaging, we restored cells to Neurobasal wtih 1 × B27, 2 mM Glutamax with 1 × primocin (in Vivogen). We used the same imaging setup as that used for immortalized cells.
We performed spot detection and quantification from FISH and live cell imaging using custom designed IDL (ITT visual information solutions) and MATLAB (Mathworks) programs. We first detected high intensity pixels by 2D- or 3D- (when using 3D image stacks) bandpass filtering the image and then applied a threshold to the bandpassed image (we adjusted the threshold value depending on the quality of each individual image). We defined as spots the local maxima within a 2 pixel radius of the pixels above threshold. We then automatically quantified the individual spots intensities the original image. To do so, we first corrected the intensity profile in a square ROI surrounding each spot for the local background, calculated as a the linear fit of the intensity vs.
position in the pixels adjacent to the ROI (we set the ROI size as 3 times the width of the point spread function, PSF). Then we calculated the position and intensity of each spot using a 2D or 3D Gaussian mask fit of the PSF34
. In the 2D projections of the tissue FISH image stacks, we identified nuclei by either automatically thresholding or manually circling the DAPI signal. We then coarse-grained the image using 32 × 32 pixels (~ 2 × 2 μm) square bins. We counted the number of mRNA particles within each bin and computed the distance from that bin to the closest nucleus. We collected quantitative data over typically 100–150 cells.