Generation of Nestin-Bmi-1-HA-GFP transgenic mice
Full-length mouse Bmi-1
cDNA was PCR amplified from total mouse embryo cDNA and HA-tagged, then inserted into the pMIG vector immediately upstream of the IRES site. The Bmi-1-IRES-GFP
fragment was then isolated by NotI digestion and inserted into NotI digested nes1852tk/lacZ
plasmid (kindly provided by Dr. Urban Lendahl). This nes1852tk/lacZ
plasmid carried the Nestin
second intronic enhancer that has previously been shown to drive transgene expression in CNS stem and progenitor cells (Kawaguchi et al., 2001
) as well as the thymidine kinase minimal promoter (Lothian and Lendahl, 1997
). The entire sequence (~7600 bp) was then linearized with ScaI and microinjected into mouse zygotes (Hogan et al., 1994
). Founder males were bred to wild-type C57Bl/6 females. Offspring were screened by GFP phenotyping of the brain in neonatal pups, and by genotyping using PCR at later ages using the primer set: 5′-TGGACACAAAGACCTCTGTG-3′ and 5′-GGTTGTTCGATGCATTTCTGC-3′.
Isolation of CNS progenitors and flow cytometry analysis
Adult lateral ventricle subventricular zone (SVZ) cells from 2-4 month old wild type or transgenic mice were obtained by micro-dissecting the lateral walls of the lateral ventricles, then dissociating for 20 min at 37 °C in 0.025% trypsin/0.5 mM EDTA (Calbiochem; San Diego, CA) plus 0.001% DNase1 (Roche; Indiannapolis, IN). After quenching the enzymatic dissociation with staining medium (L15 medium (Invitrogen; Carlsbad, CA) supplemented with 1 mg/ml BSA (Sigma; St. Louis, MO), 10 mM HEPES (pH 7.4) and 1% penicillin/streptomycin (Invitrogen)) containing 0.014% soybean trypsin inhibitor (Sigma) and 0.001% DNase1, the cells were washed and resuspended in staining medium, triturated, filtered through nylon screen (45 μm, Sefar America; Depew, NY), counted by haemocytometer, and plated.
For flow-cytometric analysis of GFP expression in transgenic mouse brain tissue, freshly dissociated single cell suspensions from neonatal mouse cerebral cortex and cerebellum, or adult SVZ were analyzed by a FACS VantageSE flow-cytometer (Becton-Dickinson; San Jose, CA). Dead cells were excluded from analysis by DAPI staining.
Neural stem cell culture, differentiation, cell proliferation and self-renewal assays
For neurosphere culture, dissociated SVZ cells were plated at clonal density (1.3 cells/μl of culture medium) onto ultra-low binding plates (Corning, Lowell, MA). The culture medium (self-renewal medium) was based on a 5:3 mixture of DMEM-low glucose: neurobasal medium (Invitrogen) supplemented with 20 ng/ml human bFGF (R&D Systems, Minneapolis, MN), 20 ng/ml EGF (R&D Systems), 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 50 μM 2-mercaptoethanol (Sigma), 1% pen/strep (Invitrogen) and 10% chick embryo extract (prepared as described (Stemple and Anderson, 1992
)). All cultures were maintained at 37°C in 6% CO2
/balance air. Neurosphere numbers and diameters were determined on the tenth day of culture.
For neurosphere culture from single cells, P5 wild type and transgenic mouse SVZ cells were dissociated as described above. To enrich neural stem cells, the dissociated SVZ cells were stained with anti-CD24 (BD Biosciences, San Jose, CA) and anti-CD15 (clone MMA) antibodies, and single CD24−CD15hi cells were sorted into each well of 96 well ultra-low binding plates. Single cell sorting was confirmed in control plates stained with DAPI. Cultures were maintained at 37°C in 6% CO2/balance air. Neurospheres were measured and subcloned for self-renewal quantification on the tenth day of culture.
To assess the differentiation of primary neurospheres, individual neurospheres were replated onto Poly-D-Lysine (PDL, Biomedical technology, Stoughton, MN) and fibronectin (Biomedical technology) coated 48-well plates and cultured adherently in differentiation medium for 7 days before being fixed and stained for markers of neurons, oligodendrocytes, and astrocytes as previously described (Molofsky et al., 2003
). Differentiation medium was the same as self-renewal medium except that it contained no EGF, and reduced concentrations of chick embryo extract (1%) and FGF (10 ng/ml).
For cell proliferation assays, dissociated adult SVZ cells were plated onto PDL and laminin (Invitrogen) coated tissue culture plates at clonal density (0.7 cells/μl) and cultured for 6 days, then pulse-labeled with 10 μM BrdU for 1 hour at 37°C before being fixed and stained with an antibody against BrdU as described (Molofsky et al., 2003
). Only stem cell colonies (identified by large compact colony morphology) were included in this analysis.
To assess the self-renewal potential of cultured neural stem cells, individual primary neurospheres were mechanically dissociated by trituration and then replated at clonal density (1.3 cells/μl) into non-adherent cultures. Secondary neurospheres were counted 10 days later and differentiated in the same manner as described above. Self-renewal is reported as the number of multipotent secondary neurospheres that arose per subcloned primary neurosphere.
Generation of Bmi-1-GFP-MSCV virus and infection of neural stem cells
Bmi-1 bearing retrovirus was made using a mouse stem cell virus (MSCV) vector (pMIG) which contains an internal ribosome entry site (IRES) followed by GFP (Van Parijs et al., 1999
). Full length mouse Bmi-1 cDNA was PCR amplified from perinatal mouse lateral ventricle SVZ cDNA. Hpa1 and Bgl2 sites were added to either end and the sequence was inserted into the MSCV vector 5' to the IRES GFP. Preparation of high-titer virus was carried out by calcium phosphate transfection of BOSC packaging cells (Pear et al., 1993
) with the Bmi-MSCV vector. Vesicular stomatitis virus G protein (VSV-G) plasmid was added during transfection to increase the infectivity of the packaged virus (Lee et al., 2001
). Cells were allowed to produce virus for 24-48 hours, then cell supernatants were collected, 0.2 micron filtered, and stored at −80°C or used fresh to infect cultured neural stem/progenitor cells.
Neonatal lateral ventricular zone (VZ) tissue from wild type or Ink4a-Arf deficient mice was dissected, dissociated, and cultured as described above. Cells were plated adherently at high density (20,000 cells per well of a six-well plate). After 24-48 hours of culture, viral supernatant was added for 12-24 hours. Then the culture medium was replaced for 24-48 hours, and cells were briefly trypsinized and replated at clonal density (0.7 cells/μl of media). Viral titers allowed infection of 25 to 90% of colonies.
Immunohistochemistry of tissue sections
Immunohistochemical staining of Bmi-1 on normal human brain tissue and brain tumor samples was performed on the DAKO Autostainer (DAKO, Carpinteria, CA) using DAKO Envision+ and diaminobenzadine (DAB) as the chromogen. De-paraffinized sections of formalin fixed tissue at 5μm thickness were labeled with mouse monoclonal anti-Bmi-1 (1:400, Clone F6, Upstate Chemicals, Pickens, SC) for 60 minutes, after microwave citric acid epitope retrieval. Appropriate negative (no primary antibody) and positive controls (high grade brain tumor) were stained in parallel with each set of tumors studied. The immunoreactivity was scored by a three-tier (negative, low (+) and high (++) positive) grading scheme.
For immunofluorescence staining of mouse brain sections, whole embryos or postnatal brains were fixed in 4% paraformaldehyde overnight, then cryoprotected in 30% sucrose, embedded in OCT (Sakura Finetek, Torrance, CA) and flash frozen. Twelve micron sections were cut on a Leica cryostat and air dried over night. Tissue sections were first blocked in PG/HN (5% horse serum, 5% goat serum, 1% BSA, 0.05% sodium azide and 0.05% Triton X-100 in 1x PBS) for 1 hour at room temperature (RT). Primary antibodies were diluted in PG/HN and incubated with the sections for 1 hour at RT, followed by washing then secondary antibody incubation for 1 hour at RT. Antibodies included mouse monoclonal anti-GFAP (1:500, Clone GA-5, Sigma); mouse monoclonal anti-b-III-Tubulin (1:500, Clone TUJ1, Covance); rat monoclonal anti-myelin basic protein (MBP, 1:100, Millipore, Temecula, CA), rabbit polyclonal anti-BFABP (1:2000, gift from T. Muller, Max-Delbruck-Center, Berlin, Germany), mouse monoclonal anti-Reelin (1:1000, Clone G10, Abcam), rabbit polyclonal anti-FoxP2 (1:500, Abcam) and Alexa 488 conjugated rabbit polyclonal anti-GFP (1:400, Invitrogen). Alexa Fluor conjugated secondary antibodies were purchased from Invitrogen and used at 1:500 dilution. Nuclei were visualized using DAPI (2μg/ml, Sigma) and slides were mounted with Prolong antifade solution (Invitrogen). Images were collected on a Leica TCS SP5 laser scanning confocal microscope, and multi-channel overlays were assembled in Adobe Photoshop.
For immunohistochemical staining of Bmi-1 on E15 telecephalon, 12 μm frozen sections of E15 wild type and transgenic mouse telencephalon were first treated with 3% H2O2/0.1% NaN3 for 1h at RT to quench endogenous peroxidase activity, then blocked with PG/HN before being labeled with mouse monoclonal anti-Bmi-1 (1:400, Clone F6, Upstate Chemicals) for 60 minutes at room temperature. The staining were then visualized using DAKO Envision+ and diaminobenzadine (DAB) as the chromogen.
To quantify adult SVZ proliferation, mice were injected with 100 mg/kg of 5-bromo-2-deoxyuridine (BrdU, Sigma), and sacrificed 2 hour later. Brains were fixed and embedded as described above. Twelve micron thick coronal sections were cut on a Leica cryostat. For detection of BrdU, DNA was first denatured in 2N HCl for 45 min at RT and neutralized with 0.1M Sodium Borate for 10 minutes. Sections were then pre-blocked in PG/HN for 1 hour at RT and stained with primary rat anti-BrdU antibody (1:500, Accurate Chemical, Westbury, NY) diluted in PG/HN for 1 hour at room temperature, followed by biotinylated goat-anti-rat IgG (Jackson Immunoresearch) for 30 minutes at RT. BrdU staining were then visualized with Alexa594-conjugated streptavidin (Invitrogen) and nuclei were counter-stained with DAPI. Slides were mounted using ProLong antifade solution (Invitrogen) and imaged on a Leica TCS SP5 laser scanning confocal microscope using a 63x oil immersion objective (NA=1.4).
To assess proliferation in E14.5 telencephalon, timed pregnant females were injected with a single dose of 100mg/ml BrdU 15 minutes prior to euthanization. Tissue processing and BrdU staining were then performed in a similar manner as described for adult tissue.
To quantify olfactory bulb neurogenesis, 1-2 month old mice were given a single injection of 100mg/kg of BrdU and kept on BrdU-containing drinking water (1mg/ml) for the next 7 days. They were switched to regular water for an additional 4 weeks before being sacrificed. Brains were fixed and embedded as described. Ten micron coronal sections of the olfactory bulb were cut on a Leica cryostat. Tissue sections were blocked with PG/HN, stained with anti-NeuN (1:100, Clone A60, Millipore), re-fixed with 4% PFA then stained for BrdU as described above. NeuN staining was visualized with Alexa555-conjugated goat-anti-mouse IgG1 secondary antibody (Invitrogen). BrdU staining was visualized with biotinylated goat-anti-rat IgG secondary antibody and Alexa594 conjugated streptavidin. Nuclei were counter-stained with DAPI. Slides were mounted in Prolong antifade (Molecular Probes), and imaged on a Leica TCS SP5 laser scanning confocal microscope using a 63x oil immersion objective (NA=1.4). Twenty five random fields of view (each containing 300-500 cells) spanning the full thickness of the olfactory bulb were counted in ImageJ software.
Neuron birth dates were determined by injecting timed pregnant females with a single dose of 100mg/ml 5-chloro-2-deoxyuridine (CldU, Sigma) at E17. Pups were sacrificed at P20, brains were fixed and embedded as described, and 40μm-thick floating sections were cut on a Leica cryostat. CldU staining was performed in a similar manner as described for BrdU, using CldU-specific (1:500, Clone BU1/75, Accurate Chemical) antibody (Kiel et al., 2007
); Cux-1 was stained using a rabbit polyclonal anti-Cux1 antibody (1:50, Santa Cruz Biotechnology; Santa Cruz, CA). CldU staining was visualized by biotinylated goat-anti-rat IgG secondary antibody and Alexa594 conjugated streptavidin, and Cux-1 staining was visualized using Alexa555 conjugated goat-anti-rabbit IgG secondary antibody.
All mice analyzed were anesthetized with 2% isoflurane/air mixture throughout MRI. Mice lay prone, head first in a 7.0T Varian MR scanner (183-mm horizontal bore, Varian, Palo Alto, CA), with the body temperature maintained at 37°C using circulated heated air. A double-tuned volume radio frequency coil was used to scan the head region of the mice. Axial T2-weighted images were acquired using a fast spin-echo sequence with the following parameters: repetition time (TR)/effective echo time (TE), 4000/47.456 ms; field of view (FOV), 30×30 mm; matrix, 256×128; slice thickness, 0.5 mm; slice spacing, 0 mm; number of slices, 25; and number of scans, 1 (total scan time ~1 min).