Messengermax

Capped and polyadenylated mRNAs encoding eGFP or Firefly luciferase were obtained from Ozbiosciences (codon table, UTR sequence, and ribosomal binding site are not disclosed by the supplier). To aid transfection of reporter mRNAs, three lipofection agents were individually used according to the manufacturer’s instructions. Approximately 15,000–20,000 J2 H. schachtii were soaked for 24 h for eGFP and mCherry: in 500 ng of mRNA, 3% lipofectamine RNAIMAX, MessengerMAX, or CRISPRMAX (Invitrogen), 100 mM octopamine (Thermo-Fisher), in a total volume of 50 µl (adjusted with Opti-MEM^™ I Reduced Serum Medium [Invitrogen]). For luciferase assays, 16 µg of mRNA, 12% lipofectamine RNAIMAX, and 100 mM octopamine (Thermo Fisher), were used in a total volume of 30 µl (adjusted with nuclease free M9 buffer).

DVS, OVS, and MTE mRNA were transfected into 293T cells with MessengerMax (Invitrogen). After 48h, we evaluated the expression of the three mRNAs with flow cytometry (FC), immunofluorescence (IF), and western blot (WB). For IF, cells were incubated with rabbit anti-s1 antibody (Sino Biological) and AF488 conjugated anti-Rabbit secondary antibody (Abcam), and images were taken with the machine. For FC, cells were trypsinized and incubated with rabbit anti-s1antibody (Sino Biological) and AF488 conjugated anti-Rabbit secondary antibody (Abcam). Data was acquired with C6 (BD Biosciences) and analyzed with FlowJo (BD Biosciences). For WB, cells were harvested and denatured in lysis buffer. Samples were loaded and run in 4-12% gradient SDS-PAGE gel and transferred to the PVDF membrane. PVDF membrane was incubated with mouse anti-s2 monoclonal antibody (Thermofisher, Cat# MA5-35946) and HRP conjugated anti-mouse secondary antibody (Invitrogen, Cat#62-6520). Anti-beta-Actin HRP Antibody for protein loading control was purchased from Santa Cruz Biotechnology (sc-47778 HRP).

HeLa cells were obtained from ATCC and maintained in DMEM media (10% FBS, v/v) at 37 °C supplied with 5% CO₂. One day prior to transfection, 120,000 cells/well were seeded on 6-well plates, resulting in ∼50–70% confluency on the day of transfection. Cells were transfected with 0.5 µg of mRNA using Lipofectamine 2000 or Messenger MAX™ (Invitrogen) by following manufacturer’s protocol. At various time points (2–72 h) post-transfection, cells were harvested and used for protein expression or enzymatic activity measurements. Under certain circumstances, 6 h after transfection with indicated mRNA constructs, the cells were allowed to continue incubating for 16 h in the medium supplied with 0.1% (v/v) DMSO (vehicle) or lactacystin (EMD Millipore Corp. USA) at 10 µM before they were collected for protein expression analysis.

In total, six different nonviral
vectors (3 polymer-based and 3 lipid-based) were screened in this
study for their ability to condense and deliver mRNA to MSCs. The
polymeric vectors included 25 kDa branched PEI (Sigma-Aldrich), Superfect
(Qiagen), and jetPEI (Polyplus). The lipid-based vectors included
jetMESSENGER (Polyplus), RNAiMAX (Invitrogen), and MessengerMax (Invitrogen).
All mRNA nanoparticles were formed with each of the nonviral vectors
according to the manufacturer’s instructions or according to
protocols previously described by our group such as in the case of
Superfect and branched PEI nanoparticles.^{29 (link),30 (link)} As the ratio of vector:nucleic acid can have a significant impact
on gene delivery efficiency, three different vector:mRNA (v/w) ratios
or nitrogen/phosphate (N/P) ratios (in the case of PEI) were screened
for each vector to identify optimal conditions for MSC transfection.
The N/P ratio refers to the ratio of positively charged nitrogen (N)
groups in a polymer-based vector to negatively charged phosphate groups
(P) in the nucleic acid with which it is complexed with. Details of
the ratios used as well as the complexation medium are detailed in Table 1.

HeLa and Hep3B cells were obtained from ATCC and were maintained in DMEM media (10% FBS) at 37 °C supplied with 5% CO2. One day prior to transfection, 500,000 cells/well were seeded on 6-well plates, resulting in ∼70% confluency on the day of transfection. Cells were transfected with 1 µg of mRNA using Lipofectamine 2000 or Messenger MAX^™ (Invitrogen) by following the manufacturer’s protocol. 24–48 h post-transfection, cells were harvested and used for protein expression or enzymatic activity measurement.

Hepa1-6 cells were seeded in two 10-cm dishes at 1 × 10⁶ cells per dish. Sixteen to twenty-four hours after seeding, the cells were co-transfected with 2 µg in vitro-transcribed dual FLuc^R208X-RLuc mRNA or dual FLuc^R208-RLuc and 4 µg in vitro-transcribed tSA1T5 using MessengerMax (Thermo Fisher Scientific). Cells were incubated at 37 °C overnight and collected by trypsinization. Mock-transfected cells were used as a negative control. Cells were rinsed four times with PBS and analysed with 2D nano PRM liquid chromatography–tandem mass spectrometry (LC–MS/MS) by Jade Bio.

For optimization of mRNA delivery, cells were seeded at ∼70% confluency and reverse transfected with indicated concentrations of mRNA-Cas9-HA containing 5-methoxyuridine (5moU) or mRNA-eGFP 5moU (TriLink) in MessengerMAX (Thermo Fisher Scientific) or RNAiMAX (Thermo Fisher Scientific). Samples were fixed either 6 or 24 h after transfection and processed for immunofluorescence. Cas9 expression was monitored by the intensity of the HA signal, which was quantified using Columbus 2.9.1 (Perkin Elmer). Unless stated otherwise, the concentration of mRNA-Cas9-HA was fixed at 40 ng per well of a 384-well plate. Cells were reverse transfected with mRNA-Cas9-HA coupled with 1% (vol/vol) MessengerMAX in OptiMEM for 6 h prior to crRNA transfection.