Construction of pAV.Ex1d-CMV>hNIS and pAV. Ex1d-CMV>hTPO/T2A/hNIS by gateway technology
The general strategies employed for construction and characterization of pAV.Ex1d-CMV>hNIS and pAV. Ex1d-CMV>hTPO/T2A/hNIS are summarized in and .
Figure 1 Schematic representation of the pAV.Ex1d-CMV>hNIS construction. The PCR products after hNIS amplification were used for a BP reaction, followed by bacterial transformation and sequencing verification. After the selection of positive clones, the (more ...)
Figure 2 Schematic representation of the pAV.Ex1d-CMV>hTPO/T2A/hNIS construction. The PCR products after hNISs and hTPOs amplification were used in the first step for a BP reaction, followed by bacterial transformation and sequencing verification. After (more ...)
Isolation and characterization of hNIS and hTPO cDNA
The full-length hNIS cDNA was removed from the hNIS gene (kindly provided by Dr. Biao Li at the Shanghai Jiaotong Medical University) by restriction digestion. The sequence of obtained hNIS cDNA was confirmed by sequencing analysis as compared to the hNIS gene in the GenBank (ID of hNIS: HH762189.1). In addition, the obtained hNIS cDNA was inserted into eukaryotic expression vector pcDNA followed by digestion using restriction enzyme EcoRI/HindIII. The digested hNIS cDNA was then analyzed by AGE (agarose gel electrophoresis) to confirm its size.
The hTPO cDNA was cut out from the pDNR-LIB-hTPO-M plasmid (Changsha Yingrun Biotechnology Co., China) mutated at the point of the 208th base from G to C as compared to the hTPO gene in the GenBank (ID of hTPO: BC095448.1). Similar to the characterization of hNIS cDNA, the mutated hTPO cDNA was confirmed by sequencing analysis and AGE.
PCR amplification of attB1/hNIS/attB2, attB1/hTPO/attB2, and attB1/hTPO/T2A/hNIS/attB2
PCR amplification of attB1/hNIS/attB2 (or attB1/hTPO/attB2): PCR was performed in 50 μL of assay solution containing 0.5 μL of Primer STAR™ HS DNA Polymerase (Takara Bio, Inc.), 10 μL of 5 × Primer STAR™ buffer (Mg2+ Plus), 4 μL of dNTP mixture (10 μM), primary forward and reverse primers (primer-F: attB1-Kozak-hNIS, sequence listed in ; primer-R: attB2-hNIS, sequence listed in , 1 μL of each primer (10 μM)), and 1 μL of hNIS (or hTPO) genomic cDNA. The mixture was heated at 98°C for 3 min followed by additional 30 PCR cycles (98°C for 10 s, 60°C for 10 s, and 72°C for 2 min per circle). The amplified attB1/hNIS/attB2 (attB1/hTPO/attB2) fragment was incubated at 72°C for 5 min to form the final PCR products, which were then purified by QIAquick Gel Extraction Kit (QIAGEN) and stored at -20°C until use.
Primers used in PCR amplification of attB1/hNIS/attB2, attB1/hTPO/attB2, and attB1/hTPO/T2A/hNIS/attB2.
PCR amplification of attB1/hTPO/T2A/hNIS/attB2: The basic PCR procedure was performed as described above, except for using different primers. The 5’T2A-3’hTPO and attB1-KozakhTPO primers were used to generate attB1-hTPO-T2A, while the 3’T2A-5’hNIS and attB2-hNIS primers were applied to yield attB2-hNIS-T2A. The obtained attB1-hTPO-T2A and attB2-hNIS-T2A (as genomic DNAs) then reacted with attB2-hNIS and attB1-Kozak-hTOP (as primers) to form the attB1/hTPO/T2A/hNIS/attB2 products. Afterwards, Fusion primer F and R were used to amplify the attB1/hTPO/T2A/hNIS/attB2 to provide the final products, which were then purified by QIAquick Gel Extraction Kit (QIAGEN) and stored at -20°C until use.
Recombination of attB × attP – construction of pDown-hNIS, pDown-hTPO, and pDown-hTPO/T2A/hNIS
Construction of pDown-hNIS (or pDown-hTPO): The previously amplified attB1/hNIS/attB2 (or attB1/hTPO/attB2) was inserted into the vector pDONR™/221 (Life Technologies, Inc.) by using the BP Clonase™ II Enzyme Mix (Life Technologies, Inc.). Briefly, in a 96-well plate, samples containing 200 ng of attB1/hNIS/ attB2 (or attB1/hTPO/attB2), 1 μL of BP Clonase™ II Enzyme Mix, 100 ng of pDONR™/221 plasmid, and 1–5 μL of TE buffer (pH 8.0) were incubated at 25°C for 3 h. Proteinase K (1 μg, Life Technologies, Inc.) was then added into the samples and incubated at 37°C for 30 min. The pDown-hNIS (or pDown-hTPO) products from the BP reaction (recombination of attB × attP) were directly used for bacterial transformation.
Construction of pDown-hTPO/T2A/hNIS: The procedure was performed similarly to the construction of pDown-hNIS as described above, except for using attB1/hTPO/T2A/hNIS/attB2. The pDown-hTPO/T2A/hNIS products from the BP reaction (recombination of attB × attP) were directly used for bacterial transformation.
An aliquot (2 μL) from pDown-hNIS (or pDownhTPO; or pDown-hTPO/T2A/hNIS) products was added into a vial containing One Shot® stb13™ Chemicallly Competent Cells E. coli (Life Technologies, Inc.) according to the manufacturer’s protocol. The samples were incubated in ice for 30 min. After heating at 42°C for 90 s, the samples were incubated in ice for 2 min following by adding of 300 μL of SOC medium (Life Technologies, Inc.). The samples were then incubated at 37°C for 1 h. After incubation, 100 μL of hNIS (or hTPO; or hTPO/T2A/hNIS) was seeded in low salt Luria-Bertani (LB) solid medium (containing 50 μg/mL of kanamycin, Life Technologies, Inc.) and incubated overnight at 37°C to produce a single colony. The remainder of transformation reaction was added into 150 μL of low-salt LB liquid medium (containing 50 μg/mL of kanamycin, Life Technologies, Inc.). The bacterial was cultured at 37°C overnight and then stored at -80°C until use.
Colony PCR of hNIS and hTPO bacterial clones
A single colony from transformation reaction was analyzed by PCR to verify the correct size of the inserted hNIS or hTPO. The hNIS (or hTPO) PCR was performed in a vial containing 30 μL of sample with 1.5 U Taq DNA Polymerase (Thermo Fisher Scientific, Inc.), pDONR™/221-specific forward primer (pUpDo-flank-f; sequence is listed in ) (10 μM) and reverse primer (pUpDo-flank-r; sequence is listed in ) (10 μM), dNTP mix (0.2 mM), 10× BioTherm™ reaction buffer (5 μM, 1.2 μL). A total of 29 PCR cycles (94°C for 30 s, 60°C for 30 s, and 72°C for 2 min per cycle) was preceded by heating to 94°C for 3 min and followed by 1-min incubation at 72°C. The sizes of PCR products were determined by AGE and ethidium bromide staining. Positive entry clones with pDown-hNIS (or pDown-hTPO) were selected and added into 150 μL of low-salt LB liquid medium (containing 50 μg/mL of kanamycin, Life Technologies, Inc.) and incubated overnight at 37°C. The entire culture was subject to plasmid isolation.
Primers used in sequencing of pDown-hNIS, pDown-hTPO, and pDown-hTPO/T2A/hNIS.
Identification by enzyme digestion for plasmid pDown-hTPO/T2A/hNIS
For enzyme digestion of pDown-hTPO/T2A/hNIS, 1 μg of plasmid DNA (Life Technologies, Inc.) was added directly into a mixture containing 1 μL of EcoRV (10 U), 1 uL of XhoІ (10 U), 2 μL of restriction buffer (10× buffer 3), 0.2 uL of BSA (10 mg/mL), and 13.8 uL of deionized water. The mixture was incubated at 37°C for 60 min. After digestion, the sample was mixed with gel loading buffer and applied to a gel for electrophoretic analysis.
DNA sequencing of pDown-hNIS, pDown-hTPO, and pDown-hTPO/T2A/hNIS
Sequencing of plasmid DNA was performed using a standard dideoxy sequencing approach [13
]. A sample (10 μL) containing 5μL of plasmid, 0.32 μM primers (pUpDo-flank-f, W1F, pUpDo-flank-r, and W1R; sequences are shown in ), 2 μL of BigDye® Terminator v3.1 Ready Reaction Mix (Life Technologies, Inc.), and 1 μL 5× sequencing buffer was heated at 95°C for 5 min followed by 25 cycles of extension reactions (95°C for 10 s, 50°C for 5 s, and 60°C for 90 s per cycle). After precipitating with sodium acetate and absolute ethanol, the resulting DNA was sequenced with an ABI 3730 xl Genetic Analyzer Capillary Array (Life Technologies, Inc.).
Recombination of attL×attR – construction of pAV.Ex1d-CMV>hNIS, pAV.Ex1d-CMV>hTPO, and pAV.Ex1d-CMV>hTPO/T2A/hNIS
Entry vectors were set up in an LR reaction to recombine the gene of interest into pAV.Des1d destination vectors. Samples containing 10 fmol pDown-hNIS (or pDown-hTPO; or pDown-hTPO/T2A/hNIS), 1 μL of LR Clonase™ II Enzyme Mix (Life Technologies, Inc.), pAV.Des1d destination vector (20 fmol), and 1–5 μL of TE buffer (pH 8.0) were incubated at 25°C for 16 h. Proteinase K (1 ug, Life Technologies, Inc.) was then added into the samples and incubated at 37°C for 30 min. The pAV.Ex1d-CMV>hNIS (or pAV.Ex1d-CMV>hTPO; or pAV.Ex1d-CMV>hTPO/T2A/hNIS) products from the LR reaction (recombination of attL × attR) were directly used for bacterial transformation.
Colony PCRs of pAV.Ex1d-CMV>hNIS (or pAV. Ex1d-CMV>hTPO) were performed as described above by using vector-specific primers (F: GAACCCACTGCTTACTGGCTT; R: TCGAGACCGAGGAGAGGGT) to verify successful cloning. To confirm pAV.Ex1d-CMV>hTPO/T2A/hNIS, similar enzyme digestion procedure was performed as described above except for using restriction buffer (10× buffer 4), and NdeI and BstBI as enzymes.
Packaging, amplification, concentration, and titration of the recombinant adenoviral vector carrying hNIS, or hTPO, or hTPO-T2A-hNIS
The expression vector pAV.Ex1d-CMV>hNIS (or pAV.Ex1d-CMV>hTPO; or pAV.Ex1d-CMV>hTPO/T2A/hNIS) was digested by enzyme PacI to generate linear adenoviral plasmid by QIAquick Gel Extraction Kit (QIAGEN, Inc.). The human embryonic kidney HEK293cells were seeded into a 6-well plate (2 × 105 cells/well) containing DMEM supplemented with 10% FBS, and cultured at 37°C for 18–24 h in a CO2 incubator (5% CO2) until cells grew to 80%~90% confluency. The HEK293 cells were then transfected with the linear adenoviral plasmid (containing 250 μL of Opti-MEM, 1 μg of plasmid DNA, and 3 μL of lipofectine in each well) at 37°C for 24 h. The HEK293 cells continued to be incubated with 10% FBS for 7 days until the formation of viral plaques, referred to cytopathic effects (CPE), was visible in most of the cells. Virus were collected by performing freezing (-80°C) and melting (37°C) of the cells for three rounds, followed by centrifuging (4°C) at 2000 g for 10 min. The supernatant was then sub-packaged and frozen at -80°C. The virus was named as P0.
The process of repeated freezing and melting was adopted for amplification of adenovirus in HEK293 cells to obtain desired virus amount. HEK293 cells were cultured as described above. When cells grew to 80%~90% confluency, 100 uL of P0 virus was added into each culture flask and incubated with 5% CO2
at 37°C until the CPE was visible in most of the cells. The cells and supernatant were then collected after centrifuging at 200 g for 10 min. The deposit was resuspended. After three rounds of freezing (-80°C) and melting (37°C) followed by centrifuging at 2000g for 10 min, the supernatant was sub-packaged and frozen at -80°C. The virus was named as P1. TCID50
(50% tissue culture infective dose) assay was carried out to titrate the adenovirus. The procedure was performed similarly to described previously [15
]. Finally, the titration value for the virus was calculated according to the Kärber formula (T = 10s+0.8
Cell culture of human prostate cancer PC-3 cells
Human prostate cancer PC-3 cells were grown at 37°C under an atmosphere of 5% CO2 in air as monolayer in 25 cm2 tissue culture flasks containing 5.0 mL of DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin solution, and 2 mM glutamine (Life Technologies, Inc.).
Adenovirus-mediated hNIS and hTPO gene transfer of PC-3 cells
PC-3 cells were seeded in a 24-well tissue culture plate at a concentration of 5 × 104 cells/ well. When cells confluence reached to 60–80%, the cell monolayers were transfected with the Ad-CMV-hTPO, Ad-CMV-hNIS, and Ad-CMV-hTPO-2A-hNIS (Multiplicity of Infection (MOI) = 20). Medium was replaced by fresh culture medium after 16 h and virus-infected cells were maintained for additional 48 h until use. All adenoviral infections were carried out in triplicates.
Immunofluorescence staining of hNIS and hTPO co-expression in transfected PC-3 cells
PC-3 cells were seeded in a 24-well plate 24 h before the experiment to reach a density of 1×105 cells/well on the day of infection. Cells were transfected with recombinant Ad5 virus described above (Ad-CMV-hTPO; or Ad-CMV-hNIS; or Ad-CMV-hTPO-2A-hNIS). After 48 h, cells were washed twice with PBS, and fixed for 20 min in 4% ice-cold paraformaldehyde. The cells were then washed twice with PBS and permeabilized with 0.2% Triton X-100 (in PBS) for 10 min. After two additional wash with PBS, the fixed cells were blocked with 5% BSA in PBS for 1 h at room temperature. The cells were then incubated with hNIS and hTPO antibody (mouse monoclonal anti-hNIS, 1:100, Lab Vision Co.; rabbit monoclonal anti-hTPO, 1:100, Abcam, Inc.), followed by PBS wash three times. The cells were incubated with anti-mouse and anti-rabbit fluorescein-conjugated secondary antibody (with a dilution of 1:100 in PBS, Lab Vision Co.) at 37°C for 1 h. After washing with PBS three times, the cells were directly observed by a fluorescence microscope (Olympus CKX41, Japan). The non-transfected cells served as the control.
Radioiodine (125I) uptake and efflux
I) uptake was measured to assess the function of hNIS and hTPO coexpressions in PC-3 cells according to the procedure described previously [17
I was purchased from Chengdu Gaotong Isotope Co., China. After 16 h gene transfection and additional 48 h maintenance, PC-3 cells were washed twice with 0.5 mL of HBSS buffer (supplemented with 10 uM sodium iodide and buffered with HEPES, pH 7.3). The cells were then incubated with 3.7 kBq of 125
I per well for 0, 5, 10, 20, 40, and 60 min, respectively, in the presence of 0.5 mL of HBSS buffer. After incubation, the medium containing 125
I was removed. The cells were then washed twice with ice-cold HBSS buffer and harvested with 100% ice-cold dehydrated alcohol. Cell lysates were collected and measured in a gamma counter (Xi’an Zhida Inc., China). Cell uptake data was presented as counts of radioactivity. Experiments were performed twice with triplicate wells.
The effect of 125I retention was evaluated by the 125I efflux kinetics in PC-3 cells co-expressed with hNIS and hTPO. Briefly, PC-3 cells transfected with Ad-CMV-hTPO, Ad-CMV-hNIS, Ad-CMV-hTPO-2A-hNIS, and control virus were washed twice with HBSS, then incubated with 3.7 kBq of 125I per well at 37°C for 60 min. After washing twice with ice-cold HBSS, cells were incubated in fresh HBSS buffer for additional 5, 15, 30, and 60 min. After incubation, the buffer was removed immediately and cells were harvested with 1 mL of dehydrated alcohol. Cell lysates were collected and measured in a gamma counter (Xi’an Zhida Inc., China). Cell uptake data was presented as counts of radioactivity. Experiments were performed twice with triplicate wells.
All experiments were carried out in triplicates unless otherwise indicated. Quantitative data were expressed as mean ± SD. Means were compared using one-way ANOVA and student’s t-test. P values of <0.05 were considered statistically significant.