Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: M3RNA-mCherry expression in multiple cell lines. (A) Representative fluorescence and phase contrast images of HCF and HEK cells upon mCherry mRNA transfection at indicated time periods. Scale bar = 100 μm. (B) Fluorescence of mCherry protein expression was quantified in HDF, HCF, and HEK cells. Plots indicate mean ± SEM of average fluorescence intensity (arbitrary units) at indicated time points (n = 3). ANOVA using GraphPad Prism with Tukey's multiple comparison test revealed significant differences between 48 and 144 h in HCF and HDF (****p < 0.0001), and significant expression at all time points in all three cell lines when compared to 4 h (****p < 0.0001, **p < 0.01, and *p < 0.05). (C) Representative flow cytometry plots of HCF and HEK cells upon M3RNA-mcherry transfection. (D) Percent transfection efficiency of sorted HDF, HCF, and HEK cells was using mock-transfected cells as control and transfected cells at 4 and 24 h. Results are plotted as mean ± SEM (n = 3) of three different cell lines. ANOVA using Dunnett's multiple comparison test revealed significant differences between the 4- and 24-h transfection with M3RNA-mCherry in HCF and HEK cells (**p < 0.01, ****p < 0.0001), and significant expression levels at 4 and 24 h transfection in all three cell lines when compared to mock transfected cells (****p < 0.0001). ANOVA, analysis of variance; HCF, human cardiac fibroblasts; HDF, human dermal fibroblasts; HEK, human embryonic kidney cells; M3RNA, microencapsulated modified messenger RNA; NS, not significant.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Expressing, Fluorescence, Transfection, Comparison, Flow Cytometry, Control, Modification

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: M3RNA-mCherry expression in neonatal rat primary cardiomyocytes. (A) Representative phase contrast and fluorescence images exhibiting rapid and declining mCherry expression at indicated time points within the rat primary neonatal cardiomyocytes. Scale bar = 100 μm. (B) Quantitation of mCherry protein expression in cardiomyocytes upon transfection (n = 3 with >10 images/time point) plotted as mean ± SEM average fluorescence intensity. ANOVA using GraphPad Prism with Tukey's multiple comparison test revealed significant differences between 24, 48, and 144 h (****p < 0.0001), and significant expression levels at 24 and 48 h when compared to 4-h time point (***p < 0.001, ****p < 0.001). (C) Representative flow cytometry plots of cardiomyocytes at the indicated time points posttransfection with mCherry mRNA using mock-transfected cells as control. Transfection efficiency was calculated from sets of three different experiments (n = 3). (D) Primary cardiomyocytes were co-transfected with mCherry, GFP, and FLuc mRNA and the representative image exhibiting triple transfection in the same cells is shown. Scale bar = 100 μm. * indicates the cardiac fibroblast transfection with three genes and ** represents the nontransfected cells in the same field. FLuc, firefly luciferase; GFP, green fluorescent protein.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Expressing, Fluorescence, Quantitation Assay, Transfection, Comparison, Flow Cytometry, Control, Luciferase

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Transfection of neonatal primary cardiomyocytes with M3RNA-mCherry does not alter the structural integrity of cardiomyocytes. Structural integrity of transfected cardiomyocytes was observed using SiR Actin and cardiac troponin staining of transfected cells. Note the similar troponin staining in transfected and nontransfected cells (indicated by # #) and specificity of troponin staining with cells positive for SiR Actin, but negative for troponin staining (#). Scale bar = 10 μm.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Transfection, Staining

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Transfection of neonatal primary cardiomyocytes with M3RNA-mCherry expression does not alter the functionality of cardiomyocytes as revealed by identical calcium channel currents within transfected cells. (A) Representative image of Ca2+ transients using Cal520AM dye (left) and mCherry (middle) exhibiting transfected cells and the overlay (right). Scale bar = 100 μm (B) Cal520AM-labeled primary cardiomyocytes demonstrating 3D representation of systole and diastole state (pseudo color). (C) Representative annotation of fluorescence intensity (arbitrary units) versus time for Ca2+ transients from the average fluorescence intensity of 28 transfected and 19 nontransfected cardiomyocytes showing similar rhythmic and coordinated Ca2+ transients in transfected and nontransfected cardiomyocytes within the same area.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Transfection, Expressing, Labeling, Fluorescence

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Transfection of neonatal primary cardiomyocytes with M3RNA-mCherry does not alter basic electrophysiological features of cardiomyocytes as revealed by voltage-current relationships in response to voltage-ramp stimulation in the whole cell mode of patch-clamp technique. (A) Image on the left shows the transmitted light image of the patched transfected neonatal cardiomyocytes with the corresponding image of mCherry fluorescence at 615 nm emission on the right. (B) Representative voltage-current relationships in mCherry-transfected neonatal cardiomyocytes obtained using the ramp protocol (shown on the top) revealed typical INa and ICa current components. (C) Voltage-sensitive inward currents were sensitive to tetrodotoxin (TTX, 5 μM), a selective inhibitor of voltage-gated Na+ channels (INa), and second current component, at peak value ∼0 mV membrane potential, was blocked by nifedipine, an inhibitor of voltage-dependent L-type Ca2+ channels (ICa).

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Transfection, Patch Clamp, Fluorescence, Membrane

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Graphical representation of nanoparticle. Nanoparticles (∼100 nm) act as carriers of mRNA coated with positively charged biological polymers that form complexes with negatively charged mRNA molecules due to ionic interactions.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques:

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Gene expression within heart upon direct myocardial injection of M3RNAs. (A) Representative bioluminescence images showing the time course of luciferase expression following direct injections of M3-FLuc mRNA into the anterior left ventricle wall. Mice were intubated and intracardiac injections were performed upon open chest surgery (see Materials and Methods). In vivo bioluminescence images of mice in the supine position are shown at 2, 24, 48, and 72 h postsurgery. (B) Quantitative bioluminescence levels as average radiance (photons/cm2) from three different experiments (five animals each; three FLuc M3RNA and two Vehicle only) were obtained from the regions of interest and were plotted against time. ****p < 0.0001, **p < 0.01. (C) IF analysis showing the mCherry expression following direct injections into the anterior left ventricle wall. mCherry-M3RNA was injected directly into the left ventricle and heart tissues were obtained 24 h postsurgery. Hearts were fixed, sectioned, and stained with anti-mCherry and troponin antibody. Upper panel shows the staining in mock-transfected hearts, while the bottom middle panel shows the mCherry expression in heart tissue (red channel), while the left panel shows the anti-mCherry antibody and the right panel is revealing the sarcomeric structures using troponin antibody. Note the structures positive for mCherry and anti mCherry, but negative for sarcomeric structures (indicated by #). (D) IF analysis of hearts (as in C above) showing GFP, mCherry, and FLuc expression upon direct injections of M3RNAs into the left ventricle and heart tissues was processed as in C above. Images were acquired using confocal microscope LSM780 with 3 × 3 tiles using the tiling tool to acquire bigger area with sharp images using a 40 × , 1.2 W lens. Upper panel shows the GFP, mCherry, and FLuc in green, red, and far-red channel, respectively, in mock-transfected hearts, while the bottom panel shows indicated gene expressions in heart tissue. In merged image, # represents areas where transfection of a single gene was observed. Scale bar = 50 μm. IF, immunofluorescence.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Gene Expression, Injection, Luciferase, Expressing, In Vivo, Staining, Transfection, Microscopy, Immunofluorescence

Journal: Tissue Engineering. Part A

Article Title: M 3 RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction

doi: 10.1089/ten.tea.2017.0445

Figure Lengend Snippet: Targeted mCherry mRNA within infarcted porcine heart using alginate gel (A) Angiography of the pig heart showing the induction of acute myocardial infarction. Left panel shows the distribution of arterial blood flow along the anterior portion of the left ventricle to determine proper balloon placement. Right panel shows the inflated balloon blocking completely the distal blood flow. Scale bar = 10 mm. (B) Serial intracardiac echocardiographic images document transition of the anterior wall of the left ventricle (arrow) from normal (top) to injured after 90 min of LAD occlusion (middle) to loaded with alginate gel having M3RNA (lower panel) over 10 min immediately after reperfusion (total time 100 min). Scale bar = 10 mm. (C) Pig heart removed and flushed with chilled normal saline, and sectioned was imaged to document mCherry expression surrounding the anterior wall infarcted area in most of the areas starting from the apex to the mid-papillary portion of the left ventricle. Scale bar = 10 mm. (D) Heart areas from the anterior wall infarcted region (mCherry transfected) versus remote regions (control) demonstrate significant mCherry expression on IF probing. Total number of pig studies n = 10 for alginate optimization and n = 4 for documentation of infarct targeting. Scale bar = 100 μm. LAD, left anterior descending coronary artery; LCx, left circumflex artery.

Article Snippet: Five hundred microliters of encapsulated mCherry mRNA (250 μg/pig) was prepared using nanoparticle in vivo transfection reagent (Altogen Biosystems, Las Vegas, NV) according to the manufacturer's instructions.

Techniques: Blocking Assay, Saline, Expressing, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: PepFect14 mediates the delivery of mRNA into human primary keratinocytes and in vivo

doi: 10.3389/fphar.2023.1219761

Figure Lengend Snippet: PF14-mRNA nanoparticles are with suitable characteristics for cellular delivery. ( A,B ) For DLS, nanoparticles were prepared using PF14 and four different mRNAs: mCherry, luciferase, EGFP and Cy5-mRNA at charge ratio (CR) 2:1. If indicated, nanoparticles were supplemented with PS80, chloroquine, MgCl 2 and/or CaCl 2 . The white bars represent the treatments, where only PF14 or indicated mRNAs were added to the cells. Data are representative of three technical replicates and expressed as ± SEM, n = 3, one-way ANOVA with post-hoc Šidák test was used, ns – not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: mRNAs: Anti-reverse cap analogue (ARCA) mRNA encoding enhanced green fluorescent protein (EGFP), i.e., EGFP-mRNA (5-moUTP), ARCA Cy5-labeled EGFP-mRNA (5mo-UTP), and CleanCap mCherry-mRNA (5mo-UTP) were purchased from APExBIO Technology (Houston, TX, USA), and CleanCap Fluc-mRNA, i.e., luciferase-mRNA (5moU) from TriLink Biotechnologies (San Diego, CA, USA).

Techniques: Luciferase

Journal: Frontiers in Pharmacology

Article Title: PepFect14 mediates the delivery of mRNA into human primary keratinocytes and in vivo

doi: 10.3389/fphar.2023.1219761

Figure Lengend Snippet: CaCl 2 increases the productive delivery of PF14-mRNA nanoparticles in human cell lines. Nanoparticles were formed from PF14 and luciferase-mRNA (A,B) or mCherry-mRNA (C,D) and after 15 min, the additives were added. HeLa (A, C) or HaCaT (B, D) cells were incubated with the nanoparticles for 24 h, and then the reporter protein expression was measured. Data are representative of three independent experiments, with each data point representing the mean of three replicates within a single experiment. Data were analyzed with one-way ANOVA with post-hoc Šidák test, **** p < 0.0001.

Article Snippet: mRNAs: Anti-reverse cap analogue (ARCA) mRNA encoding enhanced green fluorescent protein (EGFP), i.e., EGFP-mRNA (5-moUTP), ARCA Cy5-labeled EGFP-mRNA (5mo-UTP), and CleanCap mCherry-mRNA (5mo-UTP) were purchased from APExBIO Technology (Houston, TX, USA), and CleanCap Fluc-mRNA, i.e., luciferase-mRNA (5moU) from TriLink Biotechnologies (San Diego, CA, USA).

Techniques: Luciferase, Incubation, Expressing

Journal: Frontiers in Pharmacology

Article Title: PepFect14 mediates the delivery of mRNA into human primary keratinocytes and in vivo

doi: 10.3389/fphar.2023.1219761

Figure Lengend Snippet: PF14-mRNA nanoparticles are mainly transported via macropinocytosis or clathrin-mediated endocytosis into HeLa and HaCaT cell lines. (A, C, E) The influence of reagents blocking different routes of endocytosis on PF14-mRNA transfection in HeLa and HaCaT cells and primary human keratinocytes. (A) HeLa and (C) HaCaT cells were transfected with mCherry-mRNA and the effects were measured using flow cytometry. (E) The mean fluorescence intensity was measured in keratinocytes transfected with EGFP-mRNA using flow cytometry. (B, D, F) The effect of inhibitors on the viability of (B) HeLa cells, (D) HaCaT cells, and (F) primary human keratinocytes. (A–F) Data are representative of three independent samples ( n = 3). Data expressed as mean ± SEM, one-way ANOVA with Šidák multiple comparison test was used. Asterisks indicate statistically significant differences between two indicated datasets, ns – not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: mRNAs: Anti-reverse cap analogue (ARCA) mRNA encoding enhanced green fluorescent protein (EGFP), i.e., EGFP-mRNA (5-moUTP), ARCA Cy5-labeled EGFP-mRNA (5mo-UTP), and CleanCap mCherry-mRNA (5mo-UTP) were purchased from APExBIO Technology (Houston, TX, USA), and CleanCap Fluc-mRNA, i.e., luciferase-mRNA (5moU) from TriLink Biotechnologies (San Diego, CA, USA).

Techniques: Blocking Assay, Transfection, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Therapeutic expression of RAS Degrader RRSP in Pancreatic Cancer via Nanocarrier-mediated mRNA delivery

doi: 10.1101/2024.06.11.598439

Figure Lengend Snippet: (A) Schematic of nanocarrier chemistry. (B) Representative fluorescent images of PANC-1 cells after transfection of cells with 1 μg of egfp -mRNA alone, PPDP2 alone (1:40 w/v%) (PPDP2), egfp -mRNA (1 μg) with PPDP2 (PPDP2- egfp ) or egfp -mRNA (1 μg) with MessengerMAX Lipofectamine (Lipo- egfp ). (C) Quantification of the fraction of GFP positive cells (green) from five imaged frames are shown as a histogram. (D) Representative Western blot and ( E ) quantification of mCherry levels from PANC-1 xenografts injected with PPDP2 alone (PPDP2) or PPDP2– mCherry -mRNA (PPDP2- mCherry ) (n=3). (F) mCherry IHC staining and ( G ) quantification from PANC-1 xenografts injected as indicated ( n =4). P values were calculated using a one-way ANOVA and Dunnett’s multiple comparisons test, * p <0.05, ** p <0.01

Article Snippet: To assess expression of protein in tumors following PPDP2 synthetic nanocarrier delivery of mRNA, we tested mCherry -mRNA delivery by PPDP2 into xenograft tumors.

Techniques: Transfection, Western Blot, Injection, Immunohistochemistry

Journal: bioRxiv

Article Title: Therapeutic expression of RAS Degrader RRSP in Pancreatic Cancer via Nanocarrier-mediated mRNA delivery

doi: 10.1101/2024.06.11.598439

Figure Lengend Snippet: (A) Representative Western blot of RAS and pERK levels of PANC-1 cells transfected for 24 h with either vehicle only, PPDP2, PPDP2 nanocarriers loaded with rrsp -mRNA (PPDP2- rrsp ) or rrsp -mRNA mixed with MessengerMAX lipofectamine (Lipo- rrsp ) as indicated. (B,C) Densitometry quantification from replicate Western blots for RAS and phospho-ERK ( n =5). (D) Spectrophotometer quantification ( n =3) and representative image of crystal violet-stained colonies from PANC-1 cells treated as indicated in legend. (E,F) Representative Western blot and densitometry quantification ( n =3) of uncleaved/cleaved RAS levels of PANC-1 cells after 24 treatment with 5.5 μg PPDP2- rrsp -mRNA, indicative of dose for following mouse experiments. (G) Caspase Glo 3/7 luminescence activity of PANC-1 cells treated with PPDP2 and 5.5 μg rrsp-mRNA (PPDP2- rrsp ) or catalytically inactive rrsp*-mRNA (PPDP2 -rrsp* ) ( n =5). Data are presented as mean ± SEM. P values were calculated using a one-way ANOVA and Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To assess expression of protein in tumors following PPDP2 synthetic nanocarrier delivery of mRNA, we tested mCherry -mRNA delivery by PPDP2 into xenograft tumors.

Techniques: Western Blot, Transfection, Spectrophotometry, Staining, Activity Assay

Journal: bioRxiv

Article Title: Therapeutic expression of RAS Degrader RRSP in Pancreatic Cancer via Nanocarrier-mediated mRNA delivery

doi: 10.1101/2024.06.11.598439

Figure Lengend Snippet: (A) PANC-1 xenograft tumors were established and tumor volume measured every other day, excluding weekends. Day 0 indicates day of first treatment with 0.25 mg/kg dose of PPDP2- rrsp -mRNA (red) three times per week compared to PPDP2- rrsp *-mRNA (grey) or PPDP2 alone (blue). (B) Images of resected tumors on Day 30. (C) Tumor weight, (D) tumor volume and (E) change in volume compared to first day of treatment, normalized to 0, on Day 30. (F) Weight of mice measured every other day. Data were presented as mean ± SEM with n = 5. P values were calculated using a one-way ANOVA and Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To assess expression of protein in tumors following PPDP2 synthetic nanocarrier delivery of mRNA, we tested mCherry -mRNA delivery by PPDP2 into xenograft tumors.

Techniques:

Journal: Nucleic Acids Research

Article Title: Inhibition of cytoplasmic cap methylation identifies 5′ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5′ ends

doi: 10.1093/nar/gkaa046

Figure Lengend Snippet: Differential expression of TOP mRNAs in response to inhibition of cytoplasmic cap methylation. ( A ) A heat map is shown for TOP mRNAs identified as changing in the five replicate libraries prepared after culturing in medium without (–Dox) or with (+Dox) doxycycline to induce ΔN-RNMT expression. Ribosomal protein mRNAs are indicated on the right-hand side as RP, and non-ribosomal protein TOP mRNAs are indicated as TOP. The scale indicates z -scores for each determination. ( B ) Cytoplasmic RNA was recovered from cells carrying ΔN-RNMT prior to addition of doxycycline to the medium and at 3 and 6 h after addition. Each preparation was spiked with an equal amount of mCherry mRNA and analyzed by RT-qPCR for 18S rRNA and XRCC6, RPS4X, RPL8, RPS3, EIF3D and EIF3K mRNA. The results were normalized to mCherry and are shown as the mean ± standard deviation ( n = 3). * P < 0.05 by unpaired two-tailed t -test.

Article Snippet: 0.5 μg of cytoplasmic RNA was spiked with 1 fmol of CleanCap ® mCherry mRNA (Trilink L7023) and 0.5 μl of oligo(dT) 15 primer (500 μg/ml) in a total volume of 10 μl.

Techniques: Quantitative Proteomics, Inhibition, Methylation, Expressing, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Inhibition of cytoplasmic cap methylation identifies 5′ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5′ ends

doi: 10.1093/nar/gkaa046

Figure Lengend Snippet: Positioning of 75 bp sequence tags and quantitative changes following inhibition of cytoplasmic cap methylation. ( A ) The genome locations of the QuantSeq REV sequence tags are shown for one cytoplasmic capping target originally identified in (VDAC3) and two non-target mRNA (STRN4, ACTB). A portion of the latter 75 bp sequence tag is shown in black with the AAUAAA poly(A) signal highlighted in red. ( B ) The QuantSeq REV data from are displayed by volcano plot, with transcripts that decrease with ΔN-RNMT shown in red and those that increase shown on blue.

Article Snippet: 0.5 μg of cytoplasmic RNA was spiked with 1 fmol of CleanCap ® mCherry mRNA (Trilink L7023) and 0.5 μl of oligo(dT) 15 primer (500 μg/ml) in a total volume of 10 μl.

Techniques: Sequencing, Inhibition, Methylation

Journal: Nucleic Acids Research

Article Title: Inhibition of cytoplasmic cap methylation identifies 5′ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5′ ends

doi: 10.1093/nar/gkaa046

Figure Lengend Snippet: Characterization of TOP mRNA 5′ ends. ( A ) A diagram is shown of the approach to mapping TOP mRNA 5′ ends using the Lexogen TeloPrime ® system. A double-stranded DNA adapter is ligated onto the cDNA using a double-strand DNA ligase provided with the kit. 5′ ends are then PCR amplified using primers downstream of the TOP sequence and a primer to the ligated adapter. The validity of this approach for identifying capped ends has been validated using capped mCherry as a positive control and 5′ triphosphate firefly luciferase mRNA and 18S rRNA as negative controls. ( B ) Cytoplasmic RNA from uninduced (–) and 24 h doxycycline-treated cells (+) was spiked with capped mCherry mRNA, reverse transcribed, ligated to the double-strand adapter and PCR amplified and separated on a 6% native polyacrylamide gel that was then stained with SYBR Gold. The cycle numbers for each gene were: RPS3, 15 cycles; RPS4X and RPL8, 18 cycles; EIF3K, 19 cycles; mCherry and EIF3D, 22 cycles. ( C ) The PCR amplified products from uninduced cells were recovered from the gel. The single RPS4X, RPS3 and RPL8 bands were sequenced directly. The first 50 bp of each mRNA are shown and these correspond to their respective RefSeq sequences. RPS3 and the double bands for EIF3K and EIF3D were cloned, and plasmids were sequenced from 10 clones of RPS3, 24 clones of EIF3K and 27 clones of EIF3D (parentheses). The double bands for EIF3K and EIF3D were excised in a single gel piece, the recovered DNA was cloned, and plasmids were sequenced from 24 and 27 independent clones of EIF3K and EIF3D, respectively. Capped ends were identified as the sequence appended immediately adjacent to the TeloPrime adapter sequence. TOP sequences are indicated with a box, the RefSeq sequences for the first 50 nucleotides of EIF3K and EIF3D are in bold, and the respective 5′ end truncations are showed with respect to this sequence.

Article Snippet: 0.5 μg of cytoplasmic RNA was spiked with 1 fmol of CleanCap ® mCherry mRNA (Trilink L7023) and 0.5 μl of oligo(dT) 15 primer (500 μg/ml) in a total volume of 10 μl.

Techniques: Amplification, Sequencing, Positive Control, Luciferase, Reverse Transcription, Staining, Clone Assay

Journal: Nucleic Acids Research

Article Title: Inhibition of cytoplasmic cap methylation identifies 5′ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5′ ends

doi: 10.1093/nar/gkaa046

Figure Lengend Snippet: PANTHER analysis of mRNAs upregulated in ΔN-RNMT expressing cells

Article Snippet: 0.5 μg of cytoplasmic RNA was spiked with 1 fmol of CleanCap ® mCherry mRNA (Trilink L7023) and 0.5 μl of oligo(dT) 15 primer (500 μg/ml) in a total volume of 10 μl.

Techniques: Expressing, Ubiquitin Proteomics, Binding Assay, RNA Binding Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA Encoding a Bispecific Single Domain Antibody Construct Protects against Influenza A Virus Infection in Mice

doi: 10.1016/j.omtn.2020.04.015

Figure Lengend Snippet: Delivery of DOTAP/Cholesterol mRNA Lipid Nanoparticles via Intratracheal Instillation (A) Particle size analysis after incubating DOTAP/cholesterol mRNA lipoplexes in HEPES buffer or bronchoalveolar lavage fluid (BALF). BALB/c mice were i.t. administered with DOTAP/cholesterol particles formulated with different mRNA sequences (mRNA dose of 5 μg). (B) Graph and representative whole-body images showing expression levels of luciferase mRNA (5-methoxyuridine) in lungs of BALB/c mice measured via bioluminescence imaging at 6 h (n = 3 mice). DOTAP/cholesterol nanoparticles containing 1 mol% of the lipophilic dye DiR and packaged with mCherry mRNA (5-methoxyuridine) were i.t. administered, after which nanoparticle uptake (DiR fluorescence) and mRNA expression (mCherry protein) were evaluated in a variety of pulmonary cells subsets (n = 5 mice) at 6 and 24 h post administration. The flow cytometry gating strategy used to discriminate between pulmonary immune cell subsets can be found in the Figure S1 and was adopted from Knight et al. PBS-instilled mice serve as negative controls. (C) Graph shows nanoparticle uptake in CD45 positive cells (immune cells) and CD45 negative cells (nonimmune cells). (D) Nanoparticle uptake in a variety of pulmonary immune cells subsets, including alveolar macrophages, CD103 + dendritic cells (CD103 + DCs), granulocytes, other subsets of monocytes and macrophages that are not encompassed by the other subsets (Mono/macrophage), interstitial macrophages, and CD11b + DCs. (E) Representative flow cytometry plots of nanoparticle uptake and mCherry mRNA expression in alveolar macrophages. The mean percentage of DiR-positive and mCherry-positive cells, together with the mean fluorescence intensity of each signal, are given in each flow plot. (F) 5 μg of FcγRIV VHH-M2e VHH or FcγRIV VHH-RSVF VHH (irrelevant mRNA) formulated in DOTAP/cholesterol particles or 50 μg FcγRIV VHH-M2e VHH protein was instilled i.t. in BALB/c mice. 6, 24, or 48 h after instillation, BALF was isolated and cells were removed from the BALF and the ability of His 6 -tagged proteins to bind to M2e was investigated in a peptide ELISA (see Figure S2 ). De absolute titers were calculated using the standard curve shown in Figure S2 . Graphs show mean ± SEM (n = 3 mice per group). Statistical analyses on datasets were performed by one-way ANOVA followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared to negative control (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Article Snippet: The mRNA constructs encoding for firefly luciferase or the fluorescent mCherry protein were purchased from TriLink (San Diego, CA, USA).

Techniques: Particle Size Analysis, Expressing, Luciferase, Imaging, Fluorescence, Flow Cytometry, Isolation, Peptide ELISA, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA Encoding a Bispecific Single Domain Antibody Construct Protects against Influenza A Virus Infection in Mice

doi: 10.1016/j.omtn.2020.04.015

Figure Lengend Snippet: Inflammatory Response to Intratracheally Administered mRNA Nanoparticles (A) Graph depicts the relative distribution of each pulmonary immune cell subset as a percent of total viable cells, for PBS-treated mice, and mice that were i.t. instilled with DiR-labeled mRNA nanoparticles containing mCherry mRNA (mRNA dose of 5 μg), that were either sacrificed at 6 or 24 h post administration (n = 5 mice). (B) Serum samples collected from these mice were screened for the presence of inflammatory cytokine responses. Statistical analyses on datasets were performed by one-way ANOVA followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared to negative control (∗p < 0.05; ∗∗∗p < 0.001).

Article Snippet: The mRNA constructs encoding for firefly luciferase or the fluorescent mCherry protein were purchased from TriLink (San Diego, CA, USA).

Techniques: Labeling, Negative Control