TITLE: Transfection and validation of BK channel expressing HEK-293 cells for the study of miR-9 regulation.
AUTHORS: Katherine Cordero Padilla, Gerardo L. Alvarado Monefledt, Adriel Guevarez-Galan, Hector G Marrero Hernandez, Mario E. Lloret-Torres, Cristina Velazquez Marrero

[DESCRIPTION]
Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3’untranslated region (3’UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.


[GUIDELINES]
All cell culture procedures were performed inside a biological hood using a sterile cell culture technique. HEK-293 is a cell line exhibiting epithelial morphology that was isolated from the kidney of a human embryo commercially available through ATCC (American Type Culture Collection) a nonprofit, global biological resource center.

[STEPS]
SECTION: Cell Culture
1. Thaw cells and then plate them as follows:
SECTION: Cell Culture
1.1. Resuspend in 1mL of modified DMEM media composed of Dulbecco’s modified Eagle’s medium (D5796, Milwaukee, WI, USA) with 10% fetal bovine serum (FBS), 2.52mM HEPES; 10 units and 10µg/mL of Penicillin/Streptomycin (Pen/Strep), and 1mM Na-pyruvate.
SECTION: Cell Culture
1.2. Pipette 20µL of the resuspended solution onto 35mm
petri dishes (surface area of 8.8cm2). 
SECTION: Cell Culture
1.3. Flood with DMEM up to a final volume of 2mL.
SECTION: Cell Culture
2. Place cells in a Sanyo CO2 incubator (37.0oC/5% CO2 level) and allow them to reach full confluency replacing the media every 3-5 days.
SECTION: Cell Culture
3. Once the cells reach 70-90% confluency suction media from the plates and add Trypsin-EDTA 1:10 diluted in 1X PBS to solubilize the cells. Apply gentle shaking to the plates to allow for the separation of the cells from the plate. Add a volume of DMEM to the plate equal to the added volume of trypsin to prevent further cell lysis.
SECTION: Cell Culture
4. Pipette suspended cells onto 10mL tubes.
SECTION: Cell Culture
5. Remove 10mL tubes from the chemical hood and centrifuge at 1,380g for 60 seconds.
SECTION: Cell Culture
6. Place the tubes inside the chemical hood and aspirate the supernatant.
SECTION: Cell Culture
7. Resuspend in 1mL of DMEM.     
SECTION: Cell Culture
8. Extract 10µL of each tube and mix with equal volume of Trypan Blue Dye.
SECTION: Cell Culture
9. Add a single droplet of the Trypan Blue mixture to a counting slide and place in an automated cell counter. 
SECTION: Cell Culture
11. Once counted add the appropriate seeding density (0.3x106 cells) to the 35mm petri dishes and place them in the incubator and allow them to reach 70-90% confluence. 
SECTION: Transfection (BK ZERO isoform cDNA)
13. Once cells reach 70-90% confluence remove the plates from the incubator and place them in the cell culture hood.
SECTION: Transfection (miR-9 and miR-9 sponge)
23. Wash the plates 3 times with 1X PBS for 30 seconds. Gently swirl between washes.
SECTION: Transfection (miR-9 and miR-9 sponge)
24. Add serum-deprived DMEM (no FBS) to the plates and place them in the incubator for 24 hours.
SECTION: Transfection (miR-9 and miR-9 sponge)
25. After incubation prepare a 100µL solution of Opti-MEM reduced serum media containing 0.8µg of cDNA BK ZERO isoform vector and 2.0µL of Lipofectamine 2000 (11668027, Thermofisher, NY,
USA).
SECTION: Transfection (miR-9 and miR-9 sponge)
27. Add the mixture to each well of the 24-well plate and place them in the incubator for 24 hours to allow for transfection to occur.
SECTION: Transfection (miR-9 and miR-9 sponge)
28. After the incubation period, completely replace the transfection media with DMEM the reduced.
SECTION: Transfection (miR-9 and miR-9 sponge)
29. Wash 3 additional times with 1X PBS and place in modified DMEM for 24 hours in the incubator.
SECTION: Monoclonal Selection
30. After transfection, of BK ZERO constructs, remove cells from the incubator and wash 3 times (30 secs each) with 1X PBS. Add DMEM supplemented with 400µg/mL of G418 (Gentamicin)(Sigma Aldrich, A1720 Milwaukee, WI, USA) antibiotic.
SECTION: Monoclonal Selection
31. Replace half of the media volume with modified DMEM every 2-3 days for 10-14 days or until visually resistant colonies are visible.
SECTION: Monoclonal Selection
32. Transfer cells from the 24-well plate (as described in the cell culture section) and plate onto 96-well plate by limited dilution (to create monoclonal cell lines) as follows:
SECTION: Monoclonal Selection
32.1. In a 96-well plate pipette 12μL of cell culture media into all except A1.
SECTION: Monoclonal Selection
32.2. Pipette 200μL of the cell suspension into well A1.
SECTION: Monoclonal Selection
32.3. Pipette 100μL from well A1 to A2 and gently resuspend gently.
SECTION: Monoclonal Selection
32.4. Repeat dilutions from A2 to A3 and subsequently throughout the entire row.
SECTION: Monoclonal Selection
32.5. Then Pipette 100μL from all wells on Row A too wells on row B and resuspend gently.
SECTION: Monoclonal Selection
32.6. Repeat this process for pair of rows (B-C, C-D…).
SECTION: Monoclonal Selection
32.7. Add 100μL of media to each well.
SECTION: Monoclonal Selection
32.8. Incubate for 4 to 5 days. 
SECTION: Monoclonal Selection
33. Observe daily to ensure colonies were formed by a single cell.
SECTION: Monoclonal Selection
34. Identify wells containing colonies from single-cell origin. Allow these colonies to reach 40% confluence. Subculture these colonies individually for validation of construct genomic insertion.




 
SECTION: Electrophysiology
35. Stably transfected cells are plated in 35mm dishes and allowed to reach 30-50% confluence. Prior to experiments, dishes are gently washed 3 times with 1X PBS  to remove any remaining fetal bovine serum from media; and then placed in Normal Locke’s solution (mM):145 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, pH 7.5.
SECTION: Electrophysiology
37. Single-channel recordings are obtained in the cell-attached patch clamp mode under the following conditions:
SECTION: Immunocytohemistry (ICC)
45. For ICC cells were plated in 35mm glass bottom petri dishes (MATTEK Life Sciences,
P35G-1.5-20-C MA, USA).     
SECTION: Immunocytohemistry (ICC)
47. Cell were fixed by placing plates in a 4% paraformaldehyde (PFA) solution (Santa
Cruz Biotechnology, CAS 30525-89-4 TX, USA) for 1 hour while gently rocking.
SECTION: Immunocytohemistry (ICC)
48. Excess PFA was removed with 3 washes using ice-cold 1X PBS.
SECTION: Immunocytohemistry (ICC)
49. Cells were first incubated for 1 hour with Cholera Toxin B Alexa Fluor 594 conjugate (CtB) antibody(C22842,Thermofisher, New York, NY, USA) 864ng/mL to label GM1.
SECTION: Immunocytohemistry (ICC)
50. Wash 3 additional times with 1X PBS.
SECTION: Immunocytohemistry (ICC)
51. Following the final wash, remove all 1X PBS.
SECTION: Immunocytohemistry (ICC)
52. Add Fluoromount-G™ Mounting Medium, with DAPI 00-4959-52 (Thermo Fisher) to the dishes prior to placing a cover slip, Premium Cover Glass 12-548-B (Fisher Scientific USA).
SECTION: Immunocytohemistry (ICC)
53. Image using Nikon Instruments A1 Confocal Laser Microscope with 60X objective (Refractive Index=1.51 and Numerical Aperture=1.40) (Maintain imaging parameters between treatments). 
SECTION: Immunocytohemistry (ICC)
54. Analyze Mean intensity fluorescence and colocalization by Nikon Software NIS Elements v5.30 – Software, Automated Measurements – Module. 
SECTION: Electrophysiology Data Analysis
44. When seeking the half-action voltage (V1/2) from NPo versus voltage curves, determine the values from sigmoidal fits assuming the following: 
 
where V1/2 is the half-action voltage and V the applied voltage. The V1/2's were thus, obtained from the half height of the sigmoidal fits.

SECTION: Electrophysiology Data Analysis
43.1. Determine  NPo for each voltage using the following:
   
where N is the number of observed channels (levels), i the ith level number, and Ai the area of a Gaussian fit to the ith level’s in all-points-histogram from each voltage.
SECTION: Electrophysiology Data Analysis
43. Perform data analysis with IgorPro graphing and curve fitting software as follows:
SECTION: Electrophysiology
37.1. Internal (pipette)solution (mM): 150 k-gluconate, 1 MgCl2, 10 HEPES, PH 7.4.
SECTION: Electrophysiology
40. When using the standard bath, correct for the effective applied voltage by estimating correction deviations using the leak currents during cell-attached recordings.
SECTION: Electrophysiology
37.2. External bath solution (mM): 145 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, pH 7.5.
or
ISO K+: 147 KCL, 10 HEPES, 2 CaCl2, pH 7.5.
SECTION: Electrophysiology
41. Aquire Data using a HEKA EPC10 patch-clamp amplifier controlled by a Macintosh-based computer system equipped with Patch Master acquisition software.
SECTION: Electrophysiology
42. Sample Data at 25μs per sample and filtered at 10kHz low bandpass.
SECTION: Cell Culture
10. While the cells are being counted, add an additional 9mL of modified DMEM to the tube and gently resuspend, for a total volume of 10mL.
SECTION: Electrophysiology
36. Allow plates to stand 30 minutes before use.
SECTION: Electrophysiology
38. Transfer plates to the recording chamber and wash dishes with ISO K+ solution 3 to 5 times (30 secs each).
SECTION: Electrophysiology
39. Carefully create a patch seal of 1-3GΩ and begin recording.
SECTION: Transfection (miR-9 and miR-9 sponge)
22. Once cells reach 70-90% confluence remove the dishes from the incubator and place them in the cell culture hood.
SECTION: Transfection (BK ZERO isoform cDNA)
14. Wash the plates 3 times with 1X PBS for 30 seconds. Gently swirl between washes.
SECTION: Transfection (BK ZERO isoform cDNA)
15. Add serum-deprived DMEM (no FBS) to the plates and place them in the incubator for 24 hours.
SECTION: Transfection (BK ZERO isoform cDNA)
19. After the incubation period, completely exchange transfection media with DMEM.
SECTION: Transfection (BK ZERO isoform cDNA)
20. Wash 3 additional times with 1X PBS and place in modified DMEM for 24 hours in the incubator.
SECTION: Transfection (BK ZERO isoform cDNA)
16. After incubation prepare a 100µL solution of Opti-MEM reduced serum media containing 0.8µg of cDNA BK ZERO isoform vector and 2.0µL of Lipofectamine 2000 (11668027, Thermofisher, NY,
USA).
SECTION: Transfection (BK ZERO isoform cDNA)
18. Add the mixture to each well in the 24-well plate and place them in the incubator for 16 hours to allow for transfection to occur.
SECTION: Transfection (BK ZERO isoform cDNA)
17. Incubate mixture for 5 minutes at room temperature before use.
SECTION: Transfection (miR-9 and miR-9 sponge)
26. Incubate mixture for 5 minutes at room temperature before use.
SECTION: Transfection (BK ZERO isoform cDNA)
12. Reseed cells onto a 24-well plate and incubate until they reach 70-90% confluence.
SECTION: Transfection (miR-9 and miR-9 sponge)
21. Reseed cells onto a 24-well plate and incubate until they reach 70-90% confluence.
SECTION: Immunocytohemistry (ICC)
46. Cells were allowed to reach 50-60% confluence and then removed from the incubator and then wash 3 times with 1X PBS (30 secs each).