Hypoxia Reporter Element Assay

Materials and Reagents

1. Pipette tips
   
2. White 96 well plates (CELLSTAR^® Tissue Culture Plates, Greiner Bio One International, catalog number: 655083 )
   
3. HEK293 (293[HEK-293]; ATCC, catalog number: CRL-1573 )
   
4. pLightswitch_Prom_BMX (BMX promoter construct; SwitchGear Genomics, catalog number: S701154 )
   
5. pLightswitch_Prom control vector (SwitchGear Genomics, catalog number: S790005 )
   
6. pTK-Cluc (Cypridina TK control construct; SwitchGear Genomics, catalog number: SN0322S )
   
7. FuGENE^® HD (Promega, catalog number: E2311 )
   
8. DMEM, high glucose, pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11995065 )
   
9. QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies, catalog number: 200516 )
   
10. Terrific Broth Powder (BD, Difco^TM, catalog number: 243820 )
    
11. Glycerol (Sigma-Aldrich, catalog number: G9012 )
    
12. Poly-D-Lysine (Sigma-Aldrich, catalog number: P6407-5MG )
    
13. Opti-MEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 31985070 )
    
14. Fetal Bovine Serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 10437028 )
    
15. LightSwitch Dual Assay System (SwitchGear Genomics, catalog number: DA010 )
    
16. Poly-D-Lysine hydrobromide (MP Biomedicals, catalog number: 02150175 )
    
17. Terrific Broth (see Recipes)
    
18. 5x Poly-D-lysine Solution (see Recipes)
    
19. 1x Poly-D-lysine Solution (sterile) (see Recipes)
    
20. Growth media (see Recipes)
    

Equipment

1. Pipettes
   
2. Thermocycler (Eppendorf, model: Mastercycler^® pro S , catalog number: 950030020)
   
3. Table-top centrifuge (Eppendorf, model: 5810 R , catalog number: 022625101)
   
4. Swinging bucket rotor (Eppendorf, model: S-4-104 , catalog number: 5820755008)
   
5. High-performance centrifuge (Thermo Fisher Scientific, model: Sorvall^TM RC 6 Plus , catalog number: 36-101-0816)
   
6. Laminar flow hood (Thermo Fisher Scientific, model: 1300 Series Class II , Type A2, catalog number: 1323TS)
   
7. 37 °C, 5% CO₂ water-jacketed incubator (Thermo Fisher Scientific, model: Heracell VIOS 160i , catalog number: 51030285)
   
8. 37 °C non-humidified incubator (Labnet International, model: 311DS , catalog number: I5311-DS)
   
9. Hypoxia chamber (Coy Lab, model: O₂ Control InVitro Glove Box with no upgrades)
   Standardfeatures include:
   
   1. Humidified incubation box
      
   2. Temperature control
      
   3. Control of O₂ and CO₂ in 0.1% increments
10. Plate luminometer (Molecular Devices, model: SpectraMax i3x , with no upgrades)
    Standard features include:
    1. Read modes: Absorbance, Fluorescence, Luminescence
       
    2. Wavelength ranges include: Abs: 230-1,000 nm, Fl Ex: 250-830 nm, Fl EM 270-850 nm, Lumi: 300-850 nm

Software

1. GraphPad Prism (GraphPad Software, Version 6)
   
2. QuikChange Primer Design (https://www.genomics.agilent.com/primerDesignProgram.jsp)
   

Procedure

1. Site-directed mutagenesis of pLightswitch_Prom_BMX to mutate the putative HRE within in the BMX promoter
   1. Primers are designed to mutate 2 base pairs (ACGTG → ATATG) within the HRE of the BMX promoter using the QuikChange Primer Design website (referenced above).
      The CG → TA bp change has been described in the literature to effectively disrupt the HRE.
      
   2. pLightSwitch_Prom_BMX is mutated using the QuikChangeXLSite-Directed Mutagenesis kit per manufacturer’s protocol.
      1. Extension time: 4.5 min
         
      2. Substitute Difco^TM Terrific Broth for NZY⁺ broth
   3. Mutated plasmids are confirmed by sequencing using promoter insert sequencing primers (Forward: 5’-TCCATCAAAACAAAACGAAACAA-3’, Reverse: 5’-AGTCGAGCACGTTCATCTGCTT-3’) listed on the Switchgear Genomics website (http://switchgeargenomics.com/resources/vector-maps).
   
   
2. Hypoxia reporter assay (LightSwitch Dual Assay System Manual)
   Day 1 (Carry out under a Laminar flow hood)
   1. Coat two white 96-well plates with 1x Poly-D-lysine (50 μl/well) for 2 h at room temperature.
      1. Plate enough wells per plate to carry out in triplicate.
         
      2. Example samples:
         1. Empty vector (pLightSwitch_Prom) + pTK-Cluc
            
         2. Wild-type Bmx promoter (pLightSwitch_Prom_BMX) + pTK-Cluc
            
         3. HRE mutated BMX promoter (pLightSwitch_Prom_BMXmHRE) + pTK-Cluc 
      3. Use one plate per condition (i.e., 2 plates for comparison normoxia to hypoxia).
   2. Wash the plates once with sterile H₂O (200 μl/well); plate 5 x 10³ HEK293 cells per well in 100 μl of growth media and cultured overnight at 37 °C in 5% CO₂.
      
   
   Day 2
   Preparation of constructs and reagents
   1. Allow all reagents and plasmids to equilibrate to room temperature.
      Note: Plasmids should be purified using a kit or protocol that will remove endotoxin, such as PureYeild Plasmid System from Promega.
      
   2. Pre-warm Opti-MEM to 37 °C.
   
   Transfection (Carry out under a Laminar flow hood)
   1. Carry out transfections according to protocol details (LightSwitch Dual Assay System; link to protocol found above) for a 96-well plate with minimal changes.
      Cypridina TK control construct, pTK-Cluc: 1 ng (One nanogram is used instead of 10 ng due to luciferase saturation)
      pLightSwitch_Prom, pLightSwitch_Prom_BMX or pLightSwitch_Prom_BMXmHRE: 30 ng
      The ratio of FuGENE^® HD to plasmid: 3:1 (0.3 μl FuGENE:100 ng DNA per well)
      
   2. Briefly, the transfection protocol is:
      1. Combine reagents as indicated in the following table and mix well.
         
         
      2. Let sit at RT for 30 min.
         
      3. Gently drop onto seeded wells.
         
      4. Gently shake.
         
      5. Plates should be incubated at 37 °C for 24 h in normoxia (5% CO₂).
   
   Day 3 (Under a Laminar flow hood)
   1. Remove the transfection reagents by changing out the media on both plates.
      
   2. Transfer one plate into hypoxia (5% CO₂, 94% N₂, and 1% O₂) at 37 °C and maintain another one in normoxia.
      
   3. Incubate both plates for an additional 24 h.
      Note: Incubation time can be varied depending on the induction rate of your favorite gene due to hypoxia.
   
   Day 4
   Follow the manufacturer's instructions (LightSwitch Dual Assay System) for measuring Renilla (pLightSwitch_Prom vectors) and Cypridina (pTK-Cluc) luciferase activity.
   1. No alterations were made to the protocol detailed by SwitchGear Genomics. Plates were always measured fresh and never frozen.
      
   2. For hypoxia conditions (all done within the hypoxia chamber):
      1. Adding reagents and incubations are all done within the hypoxia chamber.
         
      2. Remove the plates from hypoxia and immediately read the luminescence on a plate reader.
   3. For normoxia conditions: Adding reagents and incubation are all done at room temperature on the bench top.

Data analysis

1. Renilla (pLightSwitch_Prom) luciferase activity is normalized to Cypridina (pTK-Cluc) luciferase activity and adjusted for background luciferase for each condition.
   
2. Data can then be graphed as relative luciferase activity or as fold change.(see Figure 1)
   Note: If fold change is determined, the comparison should be to the wild-type promoter under normoxia conditions.
   
   
   Figure 1. Example data
   

Notes

While there may be variation in transfection efficiency from experiment to experiment, the LightSwitch Dual Assay system is designed to improve experimental accuracy by reducing extraneous influences. Thus your reproducibility and variability should be minimal after the Renilla luciferase activity has been normalized to the Cypridina luciferase activity.

Recipes

1. Terrific Broth
   47.6 g Terrific Broth powder
   4 ml glycerol
   1 L H₂O
   Autoclaved at 121 °C for 15 min
   
2. 5x Poly-D-lysine Solution
   Dissolve 5 mg Poly-D-lysine in 1 L ddH₂O
   
3. 1x Poly-D-lysine Solution (Sterile)
   Dilute 200 ml 5x Poly-D-Lysine solution w with 800 ml ddH₂O and filter sterilize
   
4. Growth media
   DMEM
   10% FBS
   

Acknowledgments

We thank Navjotsingh Pabla for assistance with protocol development and helpful comments during manuscript preparation. This study was supported by the American Lebanese Syrian Associated Charities, NIH Cancer Center Support Grant P30 CA021765, and R01 CA138744 (to SDB). This study was also supported by funds from the Ohio State University Comprehensive Cancer Center, Pelotonia foundation, and NIH Cancer Center Support Grant P30 CA016058. Authors declare that there are no conflicts of interest or competing interests.

References

1. Chen, S., Jiang, X., Gewinner, C. A., Asara, J. M., Simon, N. I., Cai, C., Cantley, L. C. and Balk, S. P. (2013). Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases. Sci Signal 6(277): ra40. 
   
2. Davis, M. I., Hunt, J. P., Herrgard, S., Ciceri, P., Wodicka, L. M., Pallares, G., Hocker, M., Treiber, D. K. and Zarrinkar, P. P. (2011). Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 29(11): 1046-1051.
   
3. He, Y., Luo, Y., Tang, S., Rajantie, I., Salven, P., Heil, M., Zhang, R., Luo, D., Li, X., Chi, H., Yu, J., Carmeliet, P., Schaper, W., Sinusas, A. J., Sessa, W. C., Alitalo, K. and Min, W. (2006). Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis. J Clin Invest 116(9): 2344-2355.
   
4. van Oosterwijk, J. G., Buelow, D. R., Drenberg, C. D., Vasilyeva, A., Li, L., Shi, L., Wang, Y. D., Finkelstein, D., Shurtleff, S. A., Janke, L. J., Pounds, S., Rubnitz, J. E., Inaba, H., Pabla, N. and Baker, S. D. (2018). Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia. J Clin Invest 128(1): 369-380.