trans-Cyclohexane-1,2-diol

trans-Cyclohexane-1,2-diol (TCHD) is a transient dilator of the nuclear pore complex (NPC). By interacting with the hydrophobic core (FG nucleoporin) of the NPC, trans-Cyclohexane-1,2-diol can disrupt the NPC structure and reversibly increase the permeability of the nuclear pore, allowing macromolecules larger than 40 kDa (such as plasmid DNA) to enter the cell nucleus by passive diffusion, thereby enhancing the nuclear import efficiency of non-viral vectors. trans-Cyclohexane-1,2-diol can improve the efficiency of in vitro electrotransfection or lipid-mediated gene transfection, especially significantly increasing gene expression in differentiated airway epithelial cells^[1][2]. In Vitro:Lipid-mediated transfection experiment:
trans-Cyclohexane-1,2-diol (0.5%-2%; 1 h) dose-dependently increases Lipofectamine- and GL67A-mediated luciferase gene expression in 293T cells. Concentrations of ≥1.5% results in 80% cell protein loss, indicating significant cytotoxicity^[1].
Electroporation experiment: trans-Cyclohexane-1,2-diol (1%; 10-30 min) significantly increases GFP/Tomato gene expression in both B16F10 and CHO cells (1.5-2.5-fold increase in fluorescence intensity), and cell viability is not inhibited^[2].

Trans-Cyclohexane-1,2-diol (TCHD) In Vitro Electroporation Protocol^[2]
Materials:
(1) B16F10 mouse melanoma cells and CHO (Chinese hamster ovary) cells were cultured to 70% confluence, trypsinized and resuspended in electroporation buffer (PB: 10 mM K₂HPO₄/KH₂PO₄, 1 mM MgCl₂, 250 mM sucrose, pH 7.4, cell concentration adjusted to 5×10⁶ cells/mL.
(2) Plasmid DNA: pCMV-eGFP-C1 or pCMV-CpGfree-tdTomato, concentration 20 μg/mL (dissolved in electroporation buffer).
(3) Reagents: TCHD solution: 1% w/v (mass/volume) , dissolved in electroporation buffer, and sterilized by filtration. Fetal bovine serum (FBS), crystal violet staining solution, PBS buffer.
(4) Equipment: electroporator (such as Electrocell S20), parallel stainless steel electrodes (spacing 0.4 cm), fluorescence microscope, flow cytometer.
Operation steps
Cell preparation: Collect cells in logarithmic growth phase, wash twice with PB buffer after centrifugation, resuspend cells to 5×10⁶ cells/mL, add plasmid DNA to a final concentration of 20 μg/mL, and mix well.
Electrotransfection parameter setting:
Low field long pulse (LF-LP): 6 square wave pulses, 600 V/cm, 5 ms pulse duration, frequency 1 Hz.
High field short pulse (HF-SP): 4 square wave pulses, 1200 V/cm, 100 μs pulse duration, frequency 1 Hz. 100 μL of cell-DNA suspension was added to the electrode gap and the electric pulses were applied at room temperature.
TCHD treatment:
Immediately after the pulse: Immediately after the pulse, 20 μL of 1% TCHD solution (final concentration approximately 0.18% w/v) was added and gently mixed. After incubation for 10 minutes after the pulse, another 20 μL of 1% TCHD solution was added. Both treatments were incubated at 37°C for 10, 20, or 30 minutes, and the control group was added with an equal volume of electroporation buffer.
Cell culture:
After the incubation period, the cells were transferred to complete medium (DMEM or EMEM) containing 10% FBS, seeded in 12-well plates, and cultured at 37°C, 5% CO2 for 24 hours.
Detection and analysis:
(1) After 24 hours, cell viability was detected by crystal violet staining. The culture medium was discarded, the cells were washed with PBS, and 0.1% crystal violet solution was added for staining for 20 minutes. After dissolving in 10% acetic acid, the absorbance at 595 nm was measured.
(2) Flow cytometry is used to detect the percentage of GFP/Tomato positive cells and the mean fluorescence intensity (MFI). The cells were trypsinized, washed with PBS, and resuspended in FACS buffer (containing 2.5% FBS) and analyzed by flow cytometry.
(3) After fixing the cells, the distribution of fluorescent protein expression was observed using a fluorescence microscope (GFP excitation light 480/40 nm, emission light 527/40 nm; Tomato excitation light 560/40 nm, emission light 630/75 nm).
Key parameters and optimized TCHD concentration:
The optimal final concentration is 0.18% w/v (i.e., 1% stock solution is added at a volume ratio of 1:6). Higher concentrations (such as 2%) may cause cytotoxicity.