2.1. Plasmid construction
Plasmids pCMVInt expressing ϕC31 integrase, and pCSmI, carrying an inactive form of the integrase that contains a single base pair change in the catalytic serine have been previously described (Groth et al., 2000
; Olivares et al., 2002
). The pMax vector expressing the enhanced green fluorescent protein (eGFP) reporter gene under the cytomegalovirus (CMV) promoter was obtained from Amaxa Biosystems (Cologne, Germany). pBLB contains the ϕC31 integrase recognition site, attB
, and the firefly luciferase gene driven by CAGGS (Niwa et al., 1991
), a chimeric promoter combining the CMV immediate-early enhancer (IE) and the chicken β-actin/rabbit β-globin hybrid promoter. To create pBLB, the pNBL2 vector (Thyagarajan et al., 2006) was digested with Xho
I to remove the CMV promoter. This linearized vector was blunt ended using the large (Klenow) fragment of E. coli
DNA polymerase I (New England Biolabs, Ipswich, MA), followed by vector dephosphorylation using shrimp alkaline phosphatase (SAP; Roche, Palo Alto, CA). In parallel, the chimeric promoter coupling the CMV-IE and the chicken β-actin/rabbit β-globin promoter was excised from the pCAGGS vector (provided by C.H. Contag, Stanford University) with Sal
I and Eco
RI (New England BioLabs). The resulting product was blunt ended with Klenow and ligated with T4 DNA ligase (New England Biolabs) into the Xho
I-digested, blunt-ended, dephosphorylated pNBL2 vector. pBEB contains the ϕC31 integrase recognition site, attB
, and eGFP driven by CAGGS. To construct pBEB, the pDB2 vector (Keravala et al., 2006
) and the pBLB vector were digested simultaneously with Afl
I to remove the CMV and CAGGS promoters respectively. The CAGGS promoter was ligated with T4 DNA ligase into the Afl
I-digested pDB2 vector.
2.2. Neurosphere harvest and culture
Neurosphere-forming cells were harvested from brains of 4 week-old C57Bl/6 mice (Taconic, Hudson, NY) as described previously (Palmer et al., 1999
). These percoll-fractionated cells were plated in T-75 flasks in culture media composed of Neurobasal A Medium (Gibco, Carlsbad, CA) supplemented with 1% L-Glutamine (Gibco), 2% B-27 without retinoic acid (Gibco), and 20 ng/ml epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ). Neurospheres were passaged every 7 days by dissociating them with Hank’s-based Enzyme-free Cell Dissociation Buffer (Gibco) and replated at a density of 2 × 106
cells per flask in growth media.
2.3. Nucleofection and selection
On the 10th passage, neurospheres were dissociated using Hank’s-based Enzyme-free Cell Dissociation Buffer. Dissociated cells were washed with Hank’s Balanced Salt Solution (HBSS; Gibco), and 4 × 106 cells were nucleofected (Amaxa Biosystems) in duplicate using the Mouse Neural Stem Cell (mNSC) Nucleofector kit (Amaxa Biosystems) with programs “A-31” and “A-33”. 5 µg of the pMax plasmid was used for evaluating nucleofection. After nucleofection, the cells were pelleted, resuspended in growth media, transferred to a 12-well culture plate, and incubated at 37°C in a CO2 incubator for 24 h. For co-nucleofection, 4 × 106 mNPCs were co-nucleofected in duplicate with 2 µg of pBEB and 2 µg of either pCMVInt or pCSmI using the “A-33” program and the mNSC Nucleofector kit. The nucleofected cells were pelleted, resuspended, and incubated in culture medium at 37°C in a CO2 incubator for 24 h. Similarly, 4 × 106 mNPCs were co-nucleofected in duplicate with 3 µg of pBLB and 3 µg of either pCMVInt or pCSmI using the “A-33” program and the mNSC Nucleofector kit. The nucleofected cells were incubated in culture medium at 37°C in a CO2 incubator for 4 days, after which the cells were transferred to 60 mm plates and exposed to G418 selection (250 µg/ml Geneticin; Gibco) for 8 weeks. Every 48 h, the neurospheres were fed 50:50 conditioned: fresh media containing G418.
2.4. Detection of transgene expression
To detect eGFP expression after 24 h, the nucleofected mNPCs were dissociated using Hank’s-based Enzyme-free Cell Dissociation Buffer, centrifuged at 1,200 rpm for 5 min, and resuspended in HBSS. An aliquot of the cells was used for Fluorescence Activated Cell Sorting (FACS) analysis. To detect luciferase activity, the mNPCs were assayed at 1, 2, 7, 14, 21, 28, 35, 42, and 56 days after nucleofection using bioluminescence imaging as follows: 5 µl/ml of luciferin substrate stock (30 mg/ml, Xenogen Corp., Alameda, CA, USA) was added to the cells and assayed immediately for bioluminescence activity using the IVIS 200 imaging system (Xenogen Corp.). Signal was quantified and analyzed using Living Image, version 2.20 software (Xenogen Corp.).
2.5. Detection of sequence-specific integration by polymerase chain reaction
Evidence of sequence-specific integration was obtained from genomic DNA isolated from mNPCs that were nucleofected with pBLB and pCMVInt or pCSmI and selected with G418 for 8 weeks. Genomic DNA was purified using the DNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Integration of the donor plasmid pBLB was detected by polymerase chain reaction (PCR) at the mpsL1 site in the mouse genome by using primers attBF3: 5′-CGAAGCCGCGGTGCG-3′ and mpsL1-R2: 5′-GTAAATGTTATTGCGGCTCT-3′ as previously described (Portlock et al, 2006
). PCR products were run on an agarose gel, and the 290 bp expected band was excised, purified (QiaQuick PCR Purification Kit; Qiagen), cloned into pCR2.1-TOPO (TOPO Cloning Kit; Invitrogen, Carlsbad, CA), and sequenced.
2.6. Proliferation and differentiation analyses of mNPCs
To determine whether ϕC31 integrase-mediated integration of the luciferase marker gene influenced mNPC proliferation and differentiation, naïve or ϕC31-modified mNPCs were dissociated approximately 8 weeks after nucleofection in Hank’s-based Enzyme-free Cell Dissociation buffer. The cells were plated at a density of 1 × 105 cells/ml growth media (Neurobasal A, 1% L-Glutamine, 2% B27 and 20 ng/ml EGF) in 8-well Lab Tek chambers (250 µl per well; NUNC, Rochester, NY). Ninety-six hours after plating, the cells were fed with a 50:50 mix of conditioned: fresh media containing bromodeoxyuridine (BrdU; 5 µM final concentration; Sigma Aldrich, St. Louis, MO) to label dividing cells. Exactly 12 h after the cells were fed with BrdU-supplemented media, they were either fixed with 4% paraformaldehyde for 10 min and stored in fresh Tris-buffered saline (TBS) at 4°C until immunostained, or cultured in differentiation media. In order to promote the growth of neurons in addition to astrocytes, the cells were differentiated in a cocktail of Neurobasal A Medium containing 1% FBS (Gibco), 100 ng/ml all-trans retinoic acid (Sigma Aldrich), 1 ng/ml FGF-2 (Peprotech), 10 ng/ml NT-3 (Peprotech), and 10 ng/ml BDNF (Peprotech) for 10 days. The differentiated cells were fixed for 10 min with 4% paraformaldehyde and stored in fresh TBS at 4°C until immunostained.
The paraformaldehyde-fixed cells were gently rinsed in TBS and then blocked at room temperature for 30 min in 3% normal donkey serum (Jackson Immunoresearch, West Grove, PA). The cells were incubated overnight at 4°C in cocktails of mouse anti-luciferase to detect stable integration of pBLB (1:500; Serotec, Raleigh, NC), with goat anti-doublecortin (DCX) to detect immature neurons (1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA), or guinea pig anti-glial fibrillary acidic protein (GFAP) to detect astrocytes (1:750; Santa Cruz Biotechnology, Inc.). The primary antibody-labeled cells were incubated for 2 h at room temperature in the appropriate secondary antibodies conjugated to fluorescein isothiocynate (FITC) or cyanine 5 (Cy5), rinsed in TBS, and fixed with 4% paraformaldehyde. Next, to detect the dividing cell population, the cells were incubated in 2 M HCl for 20 min at 37°C to denature DNA. Following DNA denaturation, the cells were incubated with rat ascites anti-BrdU (1:500; Accurate Chemical, Westbury, NY) for 24 h at 4°C and secondary antibody conjugated to cyanine 3 (Cy3) for 2 h at room temperature. After rinsing, the cells were counterstained with 10 mg/ml DAPI (Sigma Aldrich) to detect nuclear DNA and cover slipped in a solution of 50% glycerol (Sigma Aldrich), 20% polyvinyl alcohol (Sigma Aldrich), and 2.5% w/v of the antifade medium 1,4-diazobicyclo[2,2,2]-octane (DABCO; Sigma Aldrich). All secondary antibodies were purchased from Jackson Immunoresearch and were used at a concentration of 1:500. Staining sets were done in duplicate.
Fluorescent samples were imaged using a Zeiss Meta 510 confocal laser scanning microscope using a multi-channel configuration and a 40X objective with a 2.3 electronic zoom factor, which produces an image representing a 1 µm × 1 µm area of the sample. For each staining set (Luc/BrdU/neuron, Luc/BrdU/astrocyte) and culture condition (proliferation v/s differentiation), 3 sites were randomly chosen for z-dimension scanning. Appropriate gain and black levels were set using control slides with secondary antibody alone.