ARC RNA and Venus-ARC RNA were prepared by in vitro transcription of linearized plasmid DNA, as previously described [18 (link),19 (link)]. CGG 0, 30, 62 and 99 RNAs were prepared by in vitro transcription from plasmid DNA containing FMR1 cDNA with 0, 30, 62 or 99 CGG repeats, respectively, obtained from Dr. Fry [13 (link)]. Plasmid DNAs were linearized at the Xho 1 site immediately downstream of the CGG repeat region. Labeled RNAs were prepared by in vitro transcription in the presence of Cy3- or Cy5-conjugated UTP (all UTPs from GE Healthcare Biosciences, Pittsburgh, PA) using T7 mScript Standard mRNA Production System, according to manufacturer's protocol (CellScript, Madison, WI) or Amplicap-Max T3 High Yield Message Maker Kit (CellScript, Madison, WI) followed by capping and polyadenylation using reagents supplied in the T7 mScript Standard mRNA Production System (CellScript, Madison, WI) or Amplicap-Max T3 High Yield Message Maker Kit (CellScript, Madison, WI). Intactness and purity of in vitro transcribed RNAs were analyzed by gel electrophoresis and fluorescence correlation spectroscopy (FCS).
T7 mscript standard mrna production system
Manufactured by CellScript
Sourced in United States
The T7 mScript Standard mRNA Production System is a laboratory equipment designed for the in vitro transcription of mRNA. It provides the necessary components, including the T7 RNA polymerase enzyme and appropriate buffers, to facilitate the synthesis of mRNA from DNA templates in a controlled and reproducible manner.
Automatically generated - may contain errors
Lab products found in correlation
T7 scribe standard rna ivt kit, by CellScript (2 mentions)
Transferman nk2, by Eppendorf (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Jds246, by Addgene (2 mentions)
Amplicap max t3 high yield message maker kit, by CellScript (1 mentions)
Cyanine3 (cy3), by GE Healthcare (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Pgem t vector, by Promega (1 mentions)
Rneasy minielute cleanup kit, by Qiagen (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Bioanalyzer, by Bio-Rad (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Megascript kit, by Thermo Fisher Scientific (1 mentions)
Megashortscript kit, by Thermo Fisher Scientific (1 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
2 mm cuvette, by Bio-Rad (1 mentions)
26 protocols using t7 mscript standard mrna production system
1
RNA Preparation by In Vitro Transcription
2
CRISPR/Cas9 Knock-in Experiment Protocol
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For CRISPR/Cas9 mediated knock-in experiments, the Cas9 expression plasmid JDS246 and sgRNA expression plasmid DR274 were obtained from Addgene. sgRNA was designed using Zifit software (http://zifit.partners.org/ZiFiT/ ), synthesized and cloned into the plasmid DR274. The targeted sequence for rbRosa26 are shown in Fig. 2A .
Cas9 mRNAs were transcribed in vitro, capped and polyadenylated using the T7 mScript™ Standard mRNA Production System (C-MSC100625, CELLSCRIPT, Madison, WI). sgRNA was in vitro transcribed by using T7-Scribe™ Standard RNA IVT Kit (C-AS3107, CELLSCRIPT). Cas9 mRNA and sgRNA were diluted in RNase-free TE buffer (1 mM Tris-Cl pH 8.0, 0.1 mM EDTA), stored in −80 °C in 10 μl aliquots, and were thawed and kept on ice before microinjection.
Cas9 mRNAs were transcribed in vitro, capped and polyadenylated using the T7 mScript™ Standard mRNA Production System (C-MSC100625, CELLSCRIPT, Madison, WI). sgRNA was in vitro transcribed by using T7-Scribe™ Standard RNA IVT Kit (C-AS3107, CELLSCRIPT). Cas9 mRNA and sgRNA were diluted in RNase-free TE buffer (1 mM Tris-Cl pH 8.0, 0.1 mM EDTA), stored in −80 °C in 10 μl aliquots, and were thawed and kept on ice before microinjection.
3
MHC-Insulin Peptide Chimeric Construct for CD8+ and CD4+ T-Cell Interaction
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The genetic construct using MHC class I-insulin peptide chimeric construct (26 (link)–28 (link)), for insulin-reactive CD8+ T-cell interaction, incorporated modified insulin B15-23 peptide LYLVCGERV/hβ2m (20 (link), 21 (link)). A non-relevant antigen construct, influenza nucleoprotein antigen (NP50-57, SDYEGRLI), was used as a control for antigen-specific recognition (26 (link)). For interaction with antigen-specific CD4+ T cells, a hybrid insulin-chromogranin A peptide (2.5HIP) sequence DLQTLALWSRMD (29 (link)) was utilized and a 2.5HIP peptide/I-Ag7 construct was made, based on a previous construct incorporating BDC2.5 mimotope peptide/I-Ag7 (30 (link)), with the 2.5HIP peptide substituted for the mimotope. The pGEMT vector for direct cloning of PCR products was from Promega (Madison, WI). Template DNA for in vitro transcription of mRNA was cloned into the pGEM4Z/GFP/A64 vector (Fishman et al, 2017) and was prepared as previously described (21 (link)). Construct mRNA was transcribed in vitro using the T7 mScript Standard mRNA Production System (Cell-Script, USA) to generate 5′-capped mRNA. Cells were washed twice with Opti-MEM medium (Gibco) and re-suspended in 200 μl of Opti-MEM containing the required amount of in vitro-transcribed mRNA (10–20 µg). Electroporation was performed with BTX Harvard Apparatus ECM830 (1 ms, 300 V) into B cells.
4
Overexpression of PIM Kinases in Th17 Cells
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The T7 promoter containing plasmids pGEM-GFP64A90 (link) (gift from Prof. R. Morgan, National Institutes of Health, National Cancer Institute, USA), pCMV6-PIM1, pCMV6-PIM2 and pCMV6-PIM3 (all from Origene) were first digested with restriction enzymes to generate linearized templates for IVT. pGEM-GFP plasmid was digested with SpeI, PIM1/PIM3 plasmids with XmaI and PIM2 plasmid with PmeI (all from Thermo Scientific). Next, IVT RNA was produced from these templates using T7 mScript Standard mRNA Production System (Cell Script), by following manufacturer’s instructions. The IVT RNA was purified using RNeasy MiniElute Cleanup Kit (Qiagen).
For the triple over-expression (TOE) experiments, four million cells were transfected with a total of 90 pmol of PIM IVT RNA (30 pmol for each of the three PIMs) or 90 pmol of control GFP RNA. Cells were rested for 18 h post-nucleofection and further cultured under Th17 conditions, as described above.
For the triple over-expression (TOE) experiments, four million cells were transfected with a total of 90 pmol of PIM IVT RNA (30 pmol for each of the three PIMs) or 90 pmol of control GFP RNA. Cells were rested for 18 h post-nucleofection and further cultured under Th17 conditions, as described above.
5
In Vitro Cas9 mRNA and sgRNA Preparation
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Cas9 mRNA was in vitro transcribed, caped and polyadenylated using the T7 mScript™ Standard mRNA Production System (C-MSC100625, CELLSCRIPT, Madison, WI). sgRNAs were in vitro transcribed by using T7-Scribe™ Standard RNA IVT Kit (C-AS3107, CELLSCRIPT). Cas9 mRNA and sgRNAs were diluted in RNase-free TE buffer (1 mM Tris-Cl pH 8.0, 0.1 mM EDTA), stored in −80 °C in 10 μl aliquots, and were thawed and kept on ice before microinjection.
6
In vitro RNA transfection for Th17 cells
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In vitro RNA for GFP and PRKCA were transcribed from respective plasmids using the T7 mScript Standard mRNA Production System (CELLSCRIPT, cat# C-MSC100265) following the manufacturer’s instructions. An Agilent Bioanalyzer or BioRad Experion was used to confirm the size of the RNA. The RNA was then Capped and polyadenylated following instructions from the T7 mScript kit. Cells were first transfected with either MIAT-LNA1 or NC-LNA and cultured under Th17 condition as described above for 48 h. Cells were re-transfected with 28 picomoles of IVT generated PRKCA or GFP (used as negative control) RNA or none (mock transfection). The cells were allowed to rest for 24 h in RPMI 1640 medium supplemented with 10% FCS, 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM L-glutamine, and 17ng/ml IL2 (Cat# 101-IL; R&D systems). Cells were then activated in Th17 polarizing media for 72 h and the cell culture supernatant was collected for IL-17A ELISA. After six hours of re-transfection with IVT RNA, the overexpression was checked by FACS (Figure S7A
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In vitro RNA for GFP and PRKCA were transcribed from respective plasmids using the T7 mScript Standard mRNA Production System (CELLSCRIPT, cat# C-MSC100265) following the manufacturer’s instructions. An Agilent Bioanalyzer or BioRad Experion was used to confirm the size of the RNA. The RNA was then Capped and polyadenylated following instructions from the T7 mScript kit. Cells were first transfected with either MIAT-LNA1 or NC-LNA and cultured under Th17 condition as described above for 48 h. Cells were re-transfected with 28 picomoles of IVT generated PRKCA or GFP (used as negative control) RNA or none (mock transfection). The cells were allowed to rest for 24 h in RPMI 1640 medium supplemented with 10% FCS, 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM L-glutamine, and 17ng/ml IL2 (Cat# 101-IL; R&D systems). Cells were then activated in Th17 polarizing media for 72 h and the cell culture supernatant was collected for IL-17A ELISA. After six hours of re-transfection with IVT RNA, the overexpression was checked by FACS (
7
In Vitro Transcription and Electroporation of mRNA into T Cells
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RNA was synthesized and electroporated as previously described.39 (link) Briefly, pGEM plasmids were linearized by digestion with SpeI. mRNA in vitro transcription was performed using the T7 mScript Standard mRNA production system (CellScript) as per the manufacturer’s instructions to obtain capped and tailed mRNA. Production was aliquoted and stored at −80°C until use. Expanded T cells were washed three times with Opti-MEM media (Gibco) and resuspended at 1 × 108 cells/mL. 10 mg mRNA was mixed with 1 × 107 T cells and moved into cuvettes for electroporation. After electroporated with 500 V for 700 μs, T cells were recovered in the R10 media with rhIL-2.
8
HBV s183-TCR mRNA Production
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Example 4
The HBV envelope s183-191 TCR construct (HBV s183-TCR) was derived from a pUC57-s183cys b2Aa vector, and sub-cloned it into the pVAX1 vector. Plasmids were propagated in and purified from E. coli using the One Shot Top10 E. coli kit (Invitrogen), purified using QIAGEN Endo Free Plasmid Maxi Kit (QIAGEN, Valencia, Calif.), and linearized using the XbaI restriction enzyme. The linearized DNA was used to produce the TCR mRNA using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, Austin, Tex.) or T7 mScript Standard mRNA Production System (Cellscript, Madison, Wis.); T7 RNA polymerase was added to start transcription; RNA was capped with Anti-Reverse Cap Analog (ARCA). Then, poly(A)-tail was added by E. coli Poly(A) Polymerase and ATP. The resulting product was concentrated by lithium chloride precipitation and re-dissolved in buffer.
9
Neoantigen-Specific TCR Reconstitution
10
In Vitro Transcription and Capping of mRNA
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