742European Journal of Nuclear Medicine and Molecular Imaging Vol. 31, No. 5, May 2004with
Nco
I and
Xba
I restriction enzymes (NEB, NH) and the
Xba
Isite was blunt ended. Then, the eGFP/FLuc cDNA was subclonedinto the retroviral vector SFG-Ntp [42] (kindly provided by Dr.Sadelain, MSKCC, NY), replacing Ntp cDNA between
Nco
I andblunt-ended
Bam
HI restriction sites. The resultant vector wasnamed SFG-GL. Then, the SFG-NES-HSV1-tk/GFP vector wastreated with
Nco
I restriction enzyme and the fragment containingNES-HSV1-tk lacking the stop codon was subcloned into the
Nco
Isite of the SFG-GL vector upstream and in frame with theeGFP/FLuc gene, resulting in SFG-NES-HSV1-tk/eGFP/FLuc(SFG-NES-TGL) vector. In a similar way, the truncated form of theHSV1-tk,
∆
45HSV1-tk, lacking the first 45 amino acids describedby us previously [18], was subcloned into the
Nco
I site of the SFG-GL vector upstream and in frame with the eGFP/FLuc gene, result-ing in SFG-
∆
45-HSV1-tk/eGFP/FLuc (SFG-45TGL) vector.For the production of the retroviral vectors for transduction of tumor cells, 10
µ
g of DNA of each vector-encoding plasmid wastransfected into the GPG29 packaging cell line using the calciumphosphate method as previously described [42]. The VSV-G-pseu-dotyped retroviral particles were used as cell-free viral stocks fortransduction.
Transduction of tumor cells.
The U87 human glioma cell line wasobtained from ATCC (Rockville, MD). U87 cells were grown asmonolayers in MEM supplemented with 10% FCS at 37°C in ahumidified atmosphere with 5% CO
2
. The in vitro transduction of U87 cells with the retroviral vectors was accomplished by expos-ing the cell monolayers to a filtered (0.45
µ
m) culture mediumobtained from the vector producer cells for 8h in the presence of polybrene (8
µ
g/ml, Sigma, MO).
Flow cytometry and fluorescent microscopy.
Retrovirally trans-duced U87 cells were grown as bulk cultures for 48h and sub-sequently sorted for positive GFP expression using FACS(FACSVantage, Becton Dickinson, CA); the 488-nm excitationbeam and 510-nm emission filters were used. Subcellular localiza-tion of the NES-HSV1-tk/GFP, eGFP/FLuc, NES-TGL and
∆
45-TGL proteins in transduced tumor cells was visualized by fluores-cence microscopy (Nikon, Osaka, Japan) using similar excitationand emission parameters.
Assessment of the integrity of different reporter proteins.
The in-tegrity of the different reporter proteins was assessed by westernblot analysis. Total protein was extracted from transduced tumorcells using the Mammalian Protein Extraction Reagent (Pierce,Rockford, IL) and a complete protease inhibitor cocktail (RocheDiagnostics GmbH, Mannheim, Germany). The lysates were cen-trifuged at 4°C for 10min at 14,000rpm/min, and protein concen-tration in the supernatants was measured with a BCA Protein As-say Kit (Pierce, Rockford, IL) using Tecan Safire (Tecan,Salzburg, Austria) at 562nm. The proteins were separated by elec-trophoresis in NuPAGE 4%–12% Bis-Tris Gel (Invitrogen, Carls-bad, CA) and transferred to the Invitrolon PVDF membrane (Invi-trogen, Carlsbad, CA) using a semi-dry XCell II Blot Module (In-vitrogen, Carlsbad, CA). Immunoblotted proteins were analyzedusing a monoclonal anti-GFP antibody (cat # 8362; Clontech, PaloAlto, CA) at a 1:1,000 dilution, anti-HSV1-TK monoclonal anti-body (kindly provided by Dr. Summers, Yale University) at 1:200dilution, or anti-luciferase monoclonal antibody (FL-Y, SerotecLtd., Kidlington, Oxford, UK). Secondary anti-mouse HRP conju-gated antibody (cat # 65-6420, Zymed, San-Francisco, CA) wasused at 1:4,000 dilution. Restore Western Blot Stripping Bufferwas used to strip the membrane. Detection was performed usingan ECL Western blot detection kit (cat # RPN 2106, AmershamPharmacia Biotech, Piscataway, NJ).
In vitro bioluminescence assay.
Cells were seeded at a concentra-tion 0.5
×
10
6
cells/well in six-well tissue culture plates.
D
-Lucife-rin (Xenogen, Stanford, CA) at a concentration of 0.5m
M
wasadded immediately prior to assay. Bioluminescence was measuredusing Luminoscan Ascent and analyzed by Ascent Software (Lab-Systems, Finland).
In vivo fluorescent imaging.
Whole-body imaging of GFP fluores-cence in mice was performed using a home-built digital opticalfluorescence imaging (OFI) system consisting of: a 150-W halo-gen light source LT-9500 (Lightools Research, Encinitas, CA)equipped with an excitation filter (480–490nm), two fiberopticguides with linear illuminators (each positioned at 45°), and anemission filter plate (505nm cut-off filter; 6
×
12cm). The GFPfluorescence was readily observed with the naked eye and the im-
Fig. 1A–E.
Schematic struc-tures of retroviral vectors formammalian expression of different reporter genes:
A
SFG-NES-HSV1-tk/GFP;
B
pLNES-HSV1-tk/GFP withfirefly luciferase expressedfrom internal CMV promoter;
C
SFG-GFP/firefly luciferase;
D
SFG-NES-TGL;
E
SFG-
∆
45-TGL with truncation of N-terminal sequence encodingthe first 45 amino acids of thenative protein
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