Editing MRNA Transfection Reagent

The linear template was transcribed in vitro to cap-1, Ψ-containing CFTR mRNA using CellScript (Madison, USA) products: INCOGNITO T7 Ψ-RNA Transcription Kit, ScriptCap m7G Capping System, and the ScriptCap 2'-O-Methyltransferase Kit; and the mRNA was polyadenylated using the CellScript A-Plus Poly(A) Polymerase Tailing Kit, including a 90 min incubation to generate 200+ base tails. The mRNA was recovered using Lithium Chloride Precipitation Solution (Thermo Fisher Scientific, Vilnius, Lithuania), quantified by Nanodrop 2000 (Thermo Fisher Scientific, Waltham, USA), analysed via bleach agarose gel electrophoresis [62], and stored in nuclease-free water at −80°C.

'''Xentry peptide: LCLRPVG'''

derived from an N-terminal region of the X-protein of the hepatitis B virus.&nbsp; Xentry permeates adherent cells using syndecan-4 as a portal for entry, and is uniquely restricted from entering syndecan-deficient, non-adherent cells, such as resting blood cells. Intravenous injection of Xentry alone or conjugated to β-galactosidase led to its delivery to most tissues in mice, except circulating blood cells

Biotinylated D-isomeric XP (amino acid sequence: lclrpvg grsqsrsryyrqrqrsrrrrrrs) and FITC-labelled protamine (amino acid sequence: rsqsrsryyrqrqrsrrrrrrs) and XP

'''Xentry protamine fusion peptide (XP)''':&nbsp;lclrpvg grsqsrsryyrqrqrsrrrrrrs

'''Protamine''':&nbsp;rsqsrsryyrqrqrsrrrrrrs

dissolved in water to 0.5 mM, and stored as single-use aliquots at −80°C. E6446 (Merck Millipore, Molsheim, France) was dissolved to 20 mM in DMSO (Sigma-Aldrich, Auckland, NZ), chloroquine diphosphate (Sigma-Aldrich, St. Louis, USA) and hydroxychloroquine sulfate (Sigma, St. Louis, USA) were dissolved to 30 mM in water, and then these reagents were aliquotted and stored at −20°C.

E6446&nbsp;inhibits Toll-like receptor (TLR)7 and 9 signaling,&nbsp; [https://aobious.com/aobious/cell-inhibition/11051-e6446.html https://aobious.com/aobious/cell-inhibition/11051-e6446.html]&nbsp;,&nbsp;$59.00 /&nbsp;mg

antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ)



Cells (1 × 104/well) were seeded in flat-bottomed, tissue culture-treated 96-well plates (BD Biosciences, Auckland, NZ) and cultured overnight. XP and mRNAs (mCherry RFP and EGFP; TriLink Biotechnologies, San Diego, USA) were diluted in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, USA) then mixed (1 μg XP:0.25 μg mRNA) and placed on a rocker at room temperature in the dark for 15 min. The solutions were then mixed with vehicle, E6446, CQ, and/or HCQ that had been diluted in cell culture media and then 100 μL of each mixture was added to the cells. The cells were then cultured for 4 h whereupon the treatment solutions were replaced with normal media and cultured for 20 h. Quantified experiments were repeated independently at least four times.

Transfection of cells with mRNA Cells were seeded at 4 x 105 cells/well in tissue culture-treated 12-well plates (BD Biosciences, Auckland, NZ) in 1 mL of culture media and cultured overnight. Transfection reagents, XP (10 μg) and Lipofectamine™ MessengerMax (3 μL; Thermo Fisher Scientific, Carlsbad, USA), and mRNA (5 μg) were separately diluted in Opti-MEM I Reduced Serum Medium to 50 μL volumes and incubated for 5–10 min at room temperature. Transfection reagent and mRNA solutions were then combined as appropriate and incubated at room temperature for 5 min. Vehicle/DMSO and E6446 were then mixed into these solutions as indicated, the solutions added dropwise to the media in the appropriate wells, and the plates rocked gently by hand to disperse the treatment solutions. The cells were then cultured for 24 h, with the treatment solutions replaced with normal culture medium after 4 h.

Transfection of cells with CFTR mRNA Cells were seeded at 4 x 105 cells/well in tissue culture-treated 12-well plates (BD Biosciences, Auckland, NZ) in 1 mL of culture media and cultured overnight. Transfection reagents, XP (10 μg) and Lipofectamine™ MessengerMax (3 μL; Thermo Fisher Scientific, Carlsbad, USA), and CFTR mRNA (5 μg) were separately diluted in Opti-MEM I Reduced Serum Medium to 50 μL volumes and incubated for 5–10 min at room temperature. Transfection reagent and mRNA solutions were then combined as appropriate and incubated at room temperature for 5 min. Vehicle/DMSO and E6446 were then mixed into these solutions as indicated, the solutions added dropwise to the media in the appropriate wells, and the plates rocked gently by hand to disperse the treatment solutions. The cells were then cultured for 24 h, with the treatment solutions replaced with normal culture medium after 4 h.

20 μM E6446 is good for mRNA transfection. Can replace CQ and HCQ (CQ and HCQ are&nbsp;toxicity in vivo)

E6446 was >5-fold more potent on a concentration basis than CQ at improving EGFP mRNA transfections by XP in  A549 ,  AGS ,  HepG2 cell lines

 XP, XP + 10 μM E6446, and XP + 20 μM E6446 respectively facilitated approximately 1, 18, and 62% of the CFTR expression facilitated by Lipofectamine MessengerMax 
[[Category:Protocols]]