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  • br Plasmid construction br The plasmid for expressing EGFP

    2019-10-07


    2.2. Plasmid construction
    The plasmid for expressing EGFP under a human survivin promoter (Psurvivin-EGFP) was constructed by replacing the human cytomega-lovirus (CMV) promoter in PEGFP-N3 (Takara Bio USA, Mountain View, CA) with the survivin promoter fragment. The human survivin pro-moter fragment was obtained from pGL3-survivin vector, which was described elsewhere (Xu et al., 2004). The CMV promoter was removed from pEGFP-N3 through the digestion with restriction enzymes Ase I and Bgl II (Thermo Fisher Scientific, Rochester, NY). PGL3-survivin vector was used as PCR template to amplify the proximal fragment of survivin promoter (303 bp) using the forward primer (5′-ATTAATGG ATTACAGGCGTGAGCCACTG-3′) and reverse primer (5′-AGATCTGCC GCCGCCGCCACC-3′). The forward and reverse primers also introduced Ase I and Bgl II sites to the fragment. After digestion with Ase I and BgI II, the PCR fragment were ligated with the linear pEGFP-N3 (without CMV promoter) to generate Psurvivin-EGFP. The newly constructed plasmid was validated by DNA sequencing.
    2.3. Fluorescent cell lines construction and culture medium
    Breast cancer cell line MCF-7 was purchased from American Tissue Culture Collection (ATCC, Manassas, VA). MCF-7 Fosfomycin calcium were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose 4.5 mg/mL, Corning, Tewksbury, MA) supplemented with 10% fetal bovine serum (Gibco® FBS, Invitrogen, Carlsbad, CA) and 1% nonessential amino acid  Journal of Biotechnology 289 (2019) 80–87
    (Corning) at 37 °C in a humidified incubator with 5% CO2. MCF-7 cells were transfected with plasmids pEGFP-N3 and Psurvivin-EGFP using the protocol of Lipofectamine 2000 (Invitrogen). Positive transgenic cells were selected with 500 μg/mL geneticin (G-418, Thermo Fisher Scientific) for 10 days. Then, highly EGFP-expressing colonies were detected using fluorescence microscopy and subcultured without G-418 selection for 10 passages to establish stable EGFP-expressing cell lines, which were isolated and verified (> 97%) by flow cytometry (FACS Calibur, B-D Biosciences, San Jose, CA). MCF-7 cells expressing EGFP under the CMV promoter and survivin promoter were named as MCF-7-CMV-EGFP and MCF-7-SP-EGFP, respectively.
    2.4. Correlation between cell number and EGFP fluorescence intensity
    MCF-7 cell suspension (30 μL) with different cell numbers (1000–100,000) were added in 3D PET fibrous scaffolds and 2D wells on a 384-well plate (BD Optilux™ Black/clear bottom, San Jose, CA), respectively. After seeding, 2 h incubation was performed for cell Fosfomycin calcium  at-tachment. Next, the 384-well plate was transferred into a fluorescent plate reader (TECAN GENiosPro™, Mannedorf, Switzerland) for fluor-escence measurement. The EGFP fluorescence intensity was propor-tional to the cell number in each well for both MCF-7-CMV-EGFP and MCF-7-SP-EGFP cultured in 2D and 3D (Fig. 1).
    2.5. Reverse transcriptase (RT)-PCR
    MCF-7 cells under YM-155 treatment (at 20 nM for 48 h) were harvested from cell culture T-flasks. Total RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The yield
    Fig. 1. Linearity between cell number and EGFP fluorescence intensity in 2D and 3D culture systems. A: MCF-7-SP-EGFP cells; B: MCF-7-CMV-EGFP cells. For the 2D data, the error bars are smaller than the symbols and thus not visible in the figure.
    and purity of isolated RNA samples were measured with NanoDrop (Thermo Fisher Scientific). One μg of RNA was then used as the tem-plate for RT-PCR by SuperScriptTM ІII One-Step Synthesis kit (Invitrogen). Survivin primers 5′-GCACCACTTCCAGGGTTTAT-3′ (for-ward) and 5′-CAGACGCTTCCTATCACT-3′ (reverse), EGFP primers 5′-GCTGACCCTGAAGTTCATCTG-3′ (forward) and 5′-CACCTTGATGCC GTTCTTCT-3′ (reverse) and GAPDH primers 5′-GCACAGTCAAGGCCG AGAAT-3′ (forward) and 5′-GCCTTCTCCATGGTGGTGAA-3′ (reverse) were used for assaying survivin, EGFP and GAPDH genes, respectively. GAPDH transcription was used as the control to normalize survivin and EGFP transcriptions in the tested cells. The RT-PCR products were analyzed by electrophoresis on 3% agarose gel. Ethidium bromide was used to stain the DNA products for visualization and imaging with a Gel Documentation System (Gel Doc 2000, Bio-Rad Laboratory, Hercules, CA). The band intensity on the gel was quantified using Image J soft-ware.