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Resveratrol-Responsive CArG Elements from the Egr-1 Promoter for the Induction of GADD45α to Arrest the G2/M Transition

  • Qiwen Shi
  • Deepak BhatiaEmail author
Protocol
  • 646 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1895)

Abstract

Suicide gene therapy is based on the introduction of a foreign gene into tumor cells to sensitize cells to treatment, to convert a nontoxic compound into a lethal drug, or to produce a cytotoxic effect. We have constructed a suicide gene therapy vector that contains resveratrol-responsive CArG elements from the Egr-1 promoter and the GADD45α open reading frame. CArG elements are utilized as a “molecular switch” to drive the expression of GADD45α. When transfected into lung cancer cells, the vector is able to express GADD45α upon resveratrol treatment, and subsequently leads to cell cycle arrest at the G2/M transition. In this chapter, we describe a detailed protocol for vector construction, transfection, cell viability assay, and cell cycle analysis.

Key words

Cancer gene therapy CArG elements Resveratrol GADD45α 

1 Introduction

Reinstitution of a tumor suppressor gene by gene transfer technology has been shown to result in cancer cell death, tumor regression, and angiogenesis inhibition [1]. Growth arrest and DNA damage-inducible 45 alpha (GADD45α) is a ubiquitously expressed protein involved in the regulation of DNA repair, cell cycle progression, senescence, and stress-induced signaling transduction, and has been identified as a tumor suppressor [2]. Deficiency in GADD45α is associated with the initiation and development of malignancy and is regarded as a “second genetic hit” in tumorigenesis [3, 4]. Importantly, upregulation of GADD45α is necessary for many anticancer agents to exhibit their proapoptotic and anti-growth effects in cancer cells [5, 6]. Therefore, we chose GADD45α as the suicide gene and hypothesized that overexpression of GADD45α leads to tumor cell growth arrest and apoptosis.

CArG elements are the serum response elements (SREs) or 10-nucleotide motifs of the consensus sequence CC(A/T)6GG in the Egr-1 promoter that are responsive to ROS generated by radiation and chemotherapy [7]. Both deletion construct containing the CArG elements in Egr-1 5′ distal enhancer region and synthetic promotes consisting of isolated CArG motifs are able to work as a “molecular switch” in the presence of radiation or chemotherapeutic agents to initiate the expression of cDNA engineered in the therapy vector [8, 9]. Resveratrol has been implicated to induce Egr-1 promoter activity and to exert antitumor effects via Egr-1 activation [10, 11]. Altogether, we assumed that resveratrol is sufficient to trigger CArG-based promoter to drive the transcription of cloned cDNA.

The suicide gene therapy vector we designed relies on the use of CArG elements as an inducible promoter to transcriptionally activate the expression of tumor suppressor gene GADD45α. When combined with resveratrol, the vector increases cellular GADD45α protein level and subsequently results in cell growth inhibition and cell cycle arrest at G2/M transition.

2 Materials

2.1 Materials for Cell Culture and Transfection

  1. 1.

    Culture medium for cell growth and maintenance: Dulbecco’s Modified Eagle Medium (DMEM ) containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin.

     
  2. 2.

    Sterile phosphate-buffered saline (PBS; 1×): dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 mL of deionized H2O. Adjust the pH to 7.4 with HCl, and then add deionized H2O to 1 L. Sterilize the solution by autoclave. Store at room temperature.

     
  3. 3.

    Trypsin: 0.25%.

     
  4. 4.

    Culture medium for plasmid transfection : Opti-MEM containing 10% FBS and 1% NEAA without antibiotics.

     

2.2 Materials for Plasmid Construction

  1. 1.

    LB/Ampicillin plates : Dissolve 40 g of LB agar (Miller) in 1 L of deionized H2O in a 2 L flask. Cover with aluminum foil and autoclaved. Add ampicillin sodium sulfate (50 mg/mL; Sigma-Aldrich) to a final concentration of 100 μg/mL when the temperature is about 50 °C. Pour approximately 10 mL into each Petri dish. Swirl to coat the plates. Allow the agar to solidify at room temperature. Seal the plates with tape. Store at 4 °C.

     
  2. 2.

    IPTG stock solution: Add deionized water to 1.2 g IPTG to 50 mL final volume. Filter-sterilize and store at 4 °C.

     
  3. 3.

    X-Gal stock solution: dissolve 100 mg of X-Gal in 2 mL of N,N′-dimethyl formamide (DMF). Cover with aluminum foil and store at −20 °C.

     
  4. 4.

    LB/Ampicillin/IPTG/X-Gal plates: Spread 100 μL of 100 mM IPTG and 20 μL of 50 mg/mL X-Gal over the surface of LB/Ampicillin plate and dry the plate for 30 min at room temperature.

     
  5. 5.

    Tris-acetate-EDTA (TAE) buffer (50×): Dissolve 242 g of Tris base in 800 mL of deionized water. Add 57.1 mL of glacial acetic acid and 100 mL of 500 mM EDTA (pH 8.0) solution, and deionzied water to a final volume of 1 L.

     
  6. 6.

    Agarose gel (1%): Dissolve 1 g of agarose in 100 mL of 1× TAE buffer.

     
  7. 7.

    0.5% GTG™ agarose gel: Dissolve 0.5 g of GTG™ agarose in 100 mL of 1× TAE buffer.

     
  8. 8.

    LB/Ampicillin broth: Dissolve 10 g of Bacto-tryptone, 5 g of Bacto-yeast extract, and 5 g of NaCl in 1 L of deionized water. Adjust pH to 7.0 with NaOH. Add ampicillin to a final concentration of 100 μg/mL.

     
  9. 9.

    10× oligo annealing buffer: Dissolve 100 μL of 1 M Tris–HCl (pH 8.0), 200 μL of 5 M NaCl, and 20 μL of 500 mM EDTA in 1 L of deionized water.

     
  10. 10.

    SOC Medium: Purchased from Promega (Wisconsin, USA).

     

3 Methods

3.1 Growth and Maintenance of Cells

A549, a human lung adenocarcinoma epithelial cell line, was passaged when confluency was reached, usually every 2–4 days.

3.2 Subcloning of Luciferase Reporter Gene into Gene Therapy Plasmid Vector pTarget

3.2.1 Cloning of Luciferase Reporter Gene from pPK-CMV-F3 Fusion Vector

  1. 1.
    Double restriction enzyme digestion of pPK-CMV-F3 fusion vector.
    1. (a)
      Combine the following reaction component at room temperature (see Note 1 ):

      Water, nuclease-free

      11 μL

      10× FastDigest green buffer

      2 μL

      pPK-CMV-F3 fusion vector (~150 ng/μL)

      5 μL

      FastDigest XhoI

      1 μL

      FastDigest NotI

      1 μL

      Total volume:

      20 μL

       
    2. (b)

      Mix gently and spin down.

       
    3. (c)

      Incubate the reaction mix at 37 °C in a thermal cycler for 10 min. Then inactivate the enzyme by heating at 80 °C for 5 min.

       
     
  2. 2.

    Run the product on 0.5% GTG™ agarose gel to separate DNA fragments (see Note 2 ).

     
  3. 3.

    Excise the luciferase reporter gene band (see Note 3 ), and purify the DNA fragment with a gel purification kit (see Note 4 ).

     

3.2.2 pTarget Vector Ligation and Transformation

  1. 1.

    Briefly centrifuge the pTarget vector and control insert DNA tubes to collect the contents at the bottom of the tube.

     
  2. 2.

    Vortex the thawed T4 DNA ligase 10× buffer before use (see Note 5 ).

     
  3. 3.
    Prepare the following ligation reaction mix (see Note 6 ):

    T4 DNA ligase 10× buffer

    1 μL

    pTarget (60 ng/μL)

    1 μL

    Control insert DNA (4 ng/μL)

    2 μL

    T4 DNA ligase (3 Weiss units/μL)

    1 μL

    Deionized water

    5 μL

    Total volume:

    10 μL

     
  1. 4.

    Incubate above mix overnight at 4 °C (see Note 7 ).

     
  2. 5.
    Transformation of the ligation mix.
    1. (a)

      Prepare LB/Ampicillin /IPTG/X-Gal plates .

       
    2. (b)

      JM109 competent cells were thawed on wet ice. Mix the cells by gently flicking the tube.

       
    3. (c)

      Transfer 50 μL of cells into a sterile 1.5 mL microcentrifuge tube, and add 2 μL of above ligation mix to the same tube. Gently flick the tube to mix cells and ligation mix, and incubate the tube on ice for 20 min.

       
    4. (d)

      Heat shock at exactly 42 °C for 45 s (see Note 8 ).

       
    5. (e)

      Immediately place the tube on ice for 2 min.

       
    6. (f)

      Add 950 μL of SOC medium and incubate in the 37 °C shaker at 225 rpm for 1.5 h.

       
    7. (g)

      Spread 100 μL of above cell-containing culture medium on agar plate prepared in step a, and let the medium dry for 10 min.

       
    8. (h)

      Incubate the plate overnight at a 37 °C incubator (see Note 9 ).

       
     
  3. 6.

    Next day, pick up single white colonies, and incubate each colony in 3 mL of LB broth containing Ampicillin in the 37 °C shaker at 225 rpm overnight.

     
  4. 7.

    Next day, isolate recombinant plasmid using minipreps DNA purification system .

     
  5. 8.
    Make glycerol stocks.
    1. (a)

      Remove 425 μL of overnight culture and add 75 μL of sterile glycerol.

       
    2. (b)

      Snap freeze in dry ice/ethanol bath.

       
    3. (c)

      Store stocks in −80 °C.

       
     

3.2.3 Cloning of Luciferase Reporter Gene into pTarget Vector

  1. 1.

    Obtain linear pTarget vector by double restriction enzyme digestion of ligated pTarget vector using XhoI and NotI as described in Subheading 3.2.1.

     
  2. 2.

    Ligate linear pTarget with luciferase reporter gene (final concentration is ~ 50 ng) using T4 ligase as described in Subheading 3.2.2.

     
  3. 3.

    Transform ligation mix into JM109 cells as described in Subheading 3.2.2 to create pT.luc.

     

3.3 Cloning of Resveratrol Responsive-Promoter from PCR Product (For Natural Promoter) or Oligonucleotides (For Synthetic Promoter) into pT.luc/pTarget

3.3.1 Cloning of Natural Egr-1 Promoter into pT.luc/pTarget

  1. 1.

    Egr-1 primers were designed to amplify 460 bp of Egr-1 promoter upstream of start site.

    E460 infusion forward primer :

    5′- ATGGCTCGACAGATCTGCTTGGAACCAGGGAGAG-3′

    E460 infusion reverse primer :

    5’-TCAACGGGGCGGGCGATCGCGGCCTCATTTGAAGGGTCTGG-3’

     
  2. 2.
    Perform the following PCR reaction:

    Temperature and time

    Number of cycles

    95 °C, 5 min

    1

    94 °C, 15 s; 53 °C, 60 s; 72 °C, 60 s

    5

    94 °C, 15 s; 60 °C, 60 s; 72 °C, 60 s

    25

    72 °C, 10 min

    1

    4 °C, ∞

     
  3. 3.

    Run the PCR product on GTG™ 0.5% agarose gel.

     
  4. 4.

    Excise the DNA band at ~460 bp and purify the fragment using Qiagen gel extraction kit.

     
  5. 5.
    Set up the following reaction to create linear pT.luc/pTarget :

    pT.luc/pTarget (400 ng/μL)

    10 μL

    10× FastDigest green buffer

    4 μL

    FastDigest Bgl II

    4 μL

    FastDigest AsiSl

    4 μL

    Deionized water

    18 μL

    Total volume:

    40 μL

     
  6. 6.

    Incubate the reaction mix in a thermal cycler at 37 °C for 15–20 min.

     
  7. 7.

    Cool and spin down the mix. Gel purify the linear vector as described in Subheading 3.2.1.

     
  8. 8.
    Prepare the following reaction to ligate promoter E460 with linear pT.luc/pTarget:

    E460 DNA (25 ng/μL)

    2 μL

    Linearized pT.luc/pTarget (~50 ng/μL)

    4.85 μL

    Deionized water

    3.15 μL

    Total volume:

    10 μL

     
  9. 9.

    Add 10 μL of ligation mix to one In-Fusion HD EcoDry pellet, and mix by pipetting up and down.

     
  10. 10.

    Incubate the reaction mix for 15 min at 37 °C, followed by 15 min at 50 °C, to construct pE460.luc/pE460, and then place on ice for transformation and plasmid purification as described in Subheading 3.2.2.

     

3.3.2 Cloning of Synthetic CArG Elements into pT.luc/pTarget

Synthetic CArG enhancers E5, E6, and E9NS were prepared using the following oligonucleotides (ODNs) [9]:

E5ODN1: 5′-[Phos]GATCT(CCTTATTTGG)5GCGAT-3’

E5ODN2: 5′-[Phos]CGC(CCAATAAAGG)5A-3’

E6ODN1: 5′-[Phos]GATCT(CCTTATTTGG)6GCGAT-3’

E6ODN2: 5′-[Phos]CGC(CCAATAAAGG)6A-3’

E9NSODN1: 5′-[Phos]GATCT(CCATATAAGG)9GCGAT-3’

E9NSODN2: 5′-[Phos]CGC(CCTTATATGG)9A-3’
  1. 1.

    ODNs were dissolved in deionized H2O to obtain 200 μM stock concentration of each ODN.

     
  2. 2.
    Set up the following reaction for each pair of ODNs:

    ODN1 (200 μM)

    5 μL

    ODN2 (200 μM)

    5 μL

    10× oligo annealing buffer

    2 μL

    Deionized water

    8 μL

    Total volume:

    20 μL

     
  3. 3.

    Incubate the reaction mix at 95 °C for 4 min. Remove the reaction mix from heating and cool it for 5–10 min at room temperature. Spin to collect the droplets. The reaction mix is now 50 μM stock of double ODN (dODN).

     
  4. 4.

    Remove 1 μL of dODN stock (50 μM) and dilute to 100 μL with deionized H2O. The mix is now 500 nM stock.

     
  5. 5.

    Dilute 500 nM stock to 10 nM working solution (1 μL of 500 nM dODN, 5 μL of 10× oligo annealing buffer and 44 μL of dH2O).

     
  6. 6.

    Linearize pT.luc/pTarget with FastDigest Bgl II and AsiSl enzymes as described in Subheading 3.3.1 (see Note 10 ).

     
  7. 7.
    Prepare the following reaction mix:

    dODN (10 nM)

    10 μL

    Linearized pT.luc/pTarget (50 ng/μL)

    1 μL

    T4 DNA ligase (3 Weiss units/μL)

    2 μL

    T4 DNA ligase 10× buffer

    2 μL

    Deionized water

    5 μL

    Total volume:

    20 μL

     
  8. 8.

    Incubate the above mix at 22 °C for 1 h to ligate dODN with linear pT.luc/pTarget to create pE5.luc/pE5, pE6.luc/pE6 and pE9NS.luc/pE9NS, respectively. Then heat the mix at 70 °C for 5 min, followed by transformation and plasmid purification as described in Subheading 3.2.2.

     

3.4 Evaluating Promoters for Resveratrol Induction by DLR Assay

  1. 1.

    Seed A549 cells in Opti-MEM® containing 10% fetal bovine serum and 1% NEAA without antibiotics at a density of 3 × 104 in 100 μL per well in 96-well plates (see Note 11 ).

     
  2. 2.
    Next day, transfect cells using Lipofectamine™ 2000 and CombiMag reagent prepared as follows (see Note 12 ):
    1. (a)

      DNA solution: 0.2 μg plasmid is diluted to 25 μL Opti-MEM.

       
    2. (b)

      Lipofectamine™ 2000 solution: Gently mix the reagent before use. Dilute 0.3 μL of Lipofectamine™ 2000 in 25 μL of Opti-MEM in a new eppendorf. Incubate for 5 min at room temperature.

       
    3. (c)

      CombiMag reagent: vortex the reagent before each use. Transfer 0.2 μL of CombiMag into a new eppendorf (do not dilute with any culture medium).

       
    4. (d)

      Complexes formation: add 175 μL of diluted Lipofectamine™ 2000 to 175 μL of dilute plasmid containing tube. Mix gently and add the 350 μL of DNA/Lipofectamine™ 2000 mixture immediately to the 1.4 μL of CombiMag containing eppendorf tube.

       
    5. (e)

      Mix gently and incubate for 20–25 min at room temperature.

       
     
  3. 3.

    Add 50 μL of complexes dropwise onto the cells growing in serum-containing culture medium and homogenize by rocking the plate back and forth (total volume per well should be ~150 μL).

     
  4. 4.

    Incubate the cells 20 min on the magnetic plate at 37 °C in a CO2 incubator, and then remove the magnetic plate (see Note 13).

     
  5. 5.

    Incubate cells at 37 °C in a 5% CO2 incubator for 36–48 h.

     
  6. 6.

    Treat cells with 100 μM resveratrol. Incubate overnight.

     
  7. 7.

    Aspirate the media and add 30 μL of M-Per lysis solution per well and incubate on rocker for 15 min.

     
  8. 8.

    Read luminescence using DLR buffers.

     

3.5 Cloning of GADD45α Gene into pTarget Containing Resveratrol -Responsive Promoter

3.5.1 Amplification of GADD45α ORF Clone

GADD45α ORF clone was amplified by high-fidelity hot-start PCR using Qiagen HotStar HiFidelity Polymerase Kit.
  1. 1.

    Activate HotStar HiFidelity DNA Polymerase at 95 °C for 5 min.

     
  2. 2.

    Thaw 5× HotStar HiFidelity PCR Buffer, primer solution and 5× Q-solution. Mix the solutions completely before use.

     
  3. 3.
    Prepare the following reaction mix (see Note 14 ).

    Components of reaction mix

    Volume

    Final concentration

    5× HotStar HiFidelity PCR buffer

    10 μL

    5× Q-solution

    10 μL

    Forward primer (10 μM)

    5 μL

    1 μM

    Reverse primer (10 μM)

    5 μL

    1 μM

    HotStar DNA polymerase

    1 μL

    2.5 units

    Water, nuclease-free

    17 μL

    GADD45α ORF clone (25 ng/μL)

    2 μL

    1 ng/μL

    Total volume:

    50 μL

     
    • GADD45α Fusion Forward Primer (see Note 15 ):

    • 5’-GGGCGAATTCGGATCCGCCACCATGACTTTGGAGGAATTCTCG-3′

    • GADD45α Fusion Reverse Primer :

    • 5’-TTGGAATTCGCGGCCGCTCACCGTTCAGGGAGATTAAT-3’

     
  4. 4.

    Perform PCR reaction as described in Subheading 3.3.1.

     

3.5.2 Cloning of GADD45α ORF into pTarget, pE460, pE5, pE6, and pE9NS

  1. 1.
    Prepare the following reaction to linearize pTarget , pE460, pE5, pE6, and pE9NS, respectively:

    10× FastDigest Green Buffer

    2 μL

    FastDigest BamH1

    1 μL

    FastDigest Not1

    1 μL

    Deionized water

    11 μL

    pTarget/pE460/pE5/pE6/pE9NS (~150 ng/μL)

    5 μL

    Total volume:

    20 μL

     
  1. 2.

    Get linear vectors as described in Subheading 3.2.1.

     
  2. 3.
    Prepare the following cloning reaction solution:

    Purified GADD45α ORF (25 ng/μL)

    2 μL

    Linear pTarget/pE460/pE5/pE6/pE9NS (10 ng/μL)

    8 μL

    Total volume:

    10 μL

     
  1. 4.

    Mix 10 μL of above cloning reaction solution with one In-Fusion HD EcoDry pellet by pipetting up and down.

     
  2. 5.

    Incubate the reaction solution for 15 min at 37 °C, followed by 15 min at 50 °C, to create pT.G45α, pE460.G45α, pE5.G45α, pE6.G45α, and pE9NS.G45α.

     
  3. 6.

    Place the vectors on ice for following transformation and plasmid purification as described in Subheading 3.3.2.

     
  4. 7.

    Follow the PCR protocol described in Subheading 3.1.3 to verify GADD45α insert (see Note 16 ).

     

3.6 Assaying Growth Inhibition of Tumor Cells Treated with Suicide Gene Therapy

3.6.1 Cell Viability After Vector Transfection and Resveratrol Induction

  1. 1.

    Seed A549 cells at a density of 1 × 104 per well in 96-well plates.

     
  2. 2.

    Next day, transfect cells with pE460.G45α, pE5.G45α, pE6.G45α, or pE9NS.G45α as described in Subheading 3.4.

     
  3. 3.

    Day 3, treat cells with or without 100 μM of resveratrol for 24 h.

     
  4. 4.

    Add 20 μL of 5 mg/mL MTT to each well, and incubate for 4 h. Discard media and add 100 μL of Dimethyl Sulfoxide (DMSO).

     
  5. 5.

    Incubate on a shaker at room temperature for 15 min and read the absorbance at 570 nm.

     

3.6.2 Cell Cycle Analysis by Flow Cytometry

  1. 1.

    Seed A549 cells at a density of 5 × 105 in 2 mL per well in 6-well plates.

     
  2. 2.

    Next day, transfect cells with pE460.G45α, pE5.G45α, pE6.G45α, or pE9NS.G45α as described in Subheading 3.4.

     
  3. 3.

    Day 3, treat cells with or without 100 μM of resveratrol for 24 h.

     
  4. 4.

    Day 4, harvest cells with 500 μL of trypsin for about 3 min, and stop trypsinization by adding 500 μL of culture medium. Transfer cells to 1.5 mL epi tubes.

     
  5. 5.

    Centrifuge the cells at 1000 rpm (100 × g), 4 °C for 5 min and discard supernatant.

     
  6. 6.

    Resuspend cell pellet in 1 mL of cold PBS and pipette cells up and down gently to wash the cells. Repeat step 5.

     
  7. 7.

    Wash the cells as described in step 6 again (see Note 17 ).

     
  8. 8.

    Add 500 μL of PBS containing 50 μg/mL PI, 50 μg/mL RNase, and 0.1% Triton X-100 and mix with cells by pipetting up and down gently. Incubate at room temperature for 15 min in the dark.

     
  9. 9.

    Perform flow cytometry using BD Accur™ C6 and analyze data by ModFit.

     

4 Notes

  1. 1.

    FastDigest enzymes bought from Thermo Scientific are able to digest DNA in 5–15 min. Any combination of restriction enzymes can work simultaneously in one reaction tube. FastDigest Green Buffer includes two tracking dyes (in a 1% agarose gel, blue dye migrates with 3–5 kb DNA fragments and yellow dye migrates faster than 10 bp DNA fragments). Therefore, the reaction product can be directly loaded on a gel for electrophoresis when FastDigest Green Buffer is used.

     
  2. 2.

    The whole targeted DNA fragment has to be recycles. In order to reduce the volume recycled gel, do not make the gel too thick, or use a wide and thin comb to make the wells. Make fresh electrophoresis buffer and gel every time to avoid DNA contamination. Use a DNA marker to find targeted DNA fragment since the size of the DNA fragment has already been known.

     
  3. 3.

    Use UV light to excise DNA band. Clean plastic wrap and blade without DNA contamination should be used in order to prevent exogenous DNA contamination.

     
  4. 4.

    If the gel is unable to dissolve, the heating time can be increased and the tube can be turned upside down several times to facilitate dissolution. Additionally, the gel can be cut into small pieces if it is too big.

     
  5. 5.

    T4 DNA Ligase 10× buffer should be aliquoted to avoid ATP degradation caused by multiple freeze-thaw cycles.

     
  6. 6.

    The molar ratio of PCR product to pTarget Vector may be optimized. Ratios from 3:1 to 1:3 are initial parameters. pTarget is 5.67 kb in size and is supplied at a concentration of 60 ng/μL. The appropriate amount of inserted PCR product can be calculated according to the equation provided in manufacturer’s protocol.

     
  7. 7.

    The minimum time for incubation is 3 h. Shorter incubation time may result in fewer colonies.

     
  8. 8.

    Do not shake during heat shock.

     
  9. 9.

    Generally, white colonies contain inserts. Longer incubations or storing plates at 4 °C after 37 °C overnight incubation may facilitate blue/white screening.

     
  10. 10.

    DNA sequencing can be conducted with PCIN2 forward primer and CMV reverse primer to confirm the insert of E5, E6, and E9NS.

    PCIN2 forward primer: 5’-CCACCTCTGACTTGAGCGTCG-3’

    CMV reverse primer: 5’-GGTTCACTAAACGAGCTCTGC-3’

     
  11. 11.

    The cells should be 90–95% confluent before transfection. Do not use medium containing antibiotics for transfection because antibiotics may cause cell death.

     
  12. 12.

    Transfection can be conducted with or without CombiMag. CombiMag can improve transfection rates. The ratio of DNA, CombiMag and Lipofectamine ™ may be optimized according to manufactures’ instruction.

     
  13. 13.

    The medium can be changed at this step in order to improve transfection efficiency and minimize potential cytotoxicity. Leave the cells on the magnetic plate, remove the culture medium, and replace it with fresh culture complete medium.

     
  14. 14.

    HotStar HiFidelity DNA polymerase is inactive at room temperature. Therefore, it is not necessary to keep reaction tube on ice.

     
  15. 15.

    The Tm of GADD45α Fusion Forward Primer is 58.3 °C and is calculated from ATG onward. The Tm of GADD45α Fusion Reverse Primer is 58.5 °C and is calculated from TCA onward. The size of PCR fragment is 531 bp.

     
  16. 16.

    Colony PCR can be performed before plasmid purification to determine whether the colony has desired plasmid . Use a sterile pipette tip to pick up a colony, stir the tip around the bottom of a PCR tube, and put the tip into a culture tube containing 3 mL of LB broth and 100 μg/mL ampicillin. Bacteria in the PCR tube are used for colony PCR and bacteria remain on the tip are used for incubation to obtain more bacteria. Add the following reaction mix into the PCR tube containing colony: 6 μL of nuclease-free water, 10 μL of 2× Taq, 2 μL of 10 μM forward primer, 2 μL of 10 μM of reverse primer. GADD45α Fusion primers are used to amplify GADD45α ORF . PCIN2 forward primer and CMV reverse primer are used to amplify natural Egr-1 promoter or synthetic CArG elements. Amplification parameters are: denaturation at 94 °C for 10 min, followed by 25 cycles of 94 °C, 30 s; 52 °C, 90 s; 72 °C, 90 s, and full extension at 72 °C for 5 min. GADD45α is ~511 bp in size. Natural Egr-1 promoter and synthetic CArG elements are ~277 bp in size.

     
  17. 17.

    Fixation can be added before proceeding cells to staining and analysis. For fixation, add 300 μL of cold PBS and pipette up and down to resuspend cells and make single cells. Then add 700 μL of cold ethanol dropwise while vortexing. Fix cells in this 70% ethanol solution for at least 30 min. The cells can remain in 70% ethanol for up to 1 week. Before staining, spin the fixed cells at 2000–2200 rpm (380–460 × g) for 10 min and wash twice with PBS. A higher speed of centrifuge is needed to prevent significant cell loss.

     

References

  1. 1.
    Heo DS (2002) Progress and limitations in cancer gene therapy. Genet Med 4(6 Suppl):52S–55SCrossRefGoogle Scholar
  2. 2.
    Tamura RE, de Vasconcellos JF, Sarkar D, Libermann TA, Fisher PB, Zerbini LF (2012) GADD45 proteins: central players in tumorigenesis. Curr Mol Med 12(5):634–651CrossRefGoogle Scholar
  3. 3.
    Hollander MC, Kovalsky O, Salvador JM, Kim KE, Patterson AD, Haines DC, Fornace AJ Jr (2001) Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency. Cancer Res 61(6):2487–2491PubMedGoogle Scholar
  4. 4.
    Hollander MC, Fornace AJ (2002) Genomic instability, centrosome amplification, cell cycle checkpoints and Gadd45a. Oncogene 21(40):6228–6233CrossRefGoogle Scholar
  5. 5.
    Jiang T, Soprano DR, Soprano KJ (2007) GADD45A is a mediator of CD437 induced apoptosis in ovarian carcinoma cells. J Cell Physiol 212(3):771–779CrossRefGoogle Scholar
  6. 6.
    Li P, Wang D, Yao H, Doret P, Hao G, Shen Q et al (2010) Coordination of PAD4 and HDAC2 in the regulation of p53-target gene expression. Oncogene 29(21):3153–3162CrossRefGoogle Scholar
  7. 7.
    Lopez CA, Kimchi ET, Mauceri HJ, Park JO, Mehta N, Murphy KT et al (2004) Chemoinducible gene therapy: a strategy to enhance doxorubicin anti-tumor activity. Mol Cancer Ther 3(9):1167–1175PubMedGoogle Scholar
  8. 8.
    Marples B, Greco O, Joiner MC, Scott SD (2002) Molecular approaches to chemo-radiotherapy. Eur J Cancer 38(2):231–239CrossRefGoogle Scholar
  9. 9.
    Scott SD, Joiner MC, Marples B (2002) Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene Ther 9(20):1396–1402CrossRefGoogle Scholar
  10. 10.
    Whitlock NC, Bahn JH, Lee S-H, Eling TE, Baek SJ (2011) Resveratrol-induced apoptosis is mediated by early growth response-1, Krüppel-like factor 4, and activating transcription factor 3. Cancer Prev Res 4(1):116–127CrossRefGoogle Scholar
  11. 11.
    Bickenbach KA, Veerapong J, Shao MY, Mauceri HJ, Posner MC, Kron SJ, Weichselbaum RR (2008) Resveratrol is an effective inducer of CArG-driven TNF-alpha gene therapy. Cancer Gene Ther 15(3):133–139CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Collaborative Innovation Center of Yangtze River Delta Region Green PharmaceuticalsZhejiang University of TechnologyHangzhouChina
  2. 2.Bernard J. Dunn School of Pharmacy, Shenandoah UniversityFairfaxUSA

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