egfp mrna  (Novoprotein)

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Workflow of polysome profiling performed in adult Drosophila heads and mouse brains. A lysate prepared from freshly separated fly heads or mouse brains in the presence of cycloheximide was subjected to ultracentrifugation in a sucrose gradient, in which the number of ribosomes bound to RNAs increases with density. RNA was isolated from each fraction for total RNA sequencing to calculate the translation status of individual mRNA isoforms, based on the expression of each transcript relative to lysate. (B) Differential translation of short or long 3’UTR isoforms for each gene in fly heads and mouse brains. Shown are genes in which the indicated 3’UTR isoforms (short or long) are enriched in the translating fractions. (C) Proportion of genes in fly heads and mouse brains that display differential translation of 3’UTR isoforms. Values indicate the number of genes for which isoform expression ratios are biased (compared to lysate) towards the short (blue) or long (red) 3’UTR isoform. (D) Heat maps representing the differential usage of proximal or distal poly(A) sites in the indicated polysome profile fractions, in 1064 Drosophila genes clustered by Ward’s method. All genes with differential translation of 3’UTR isoforms are shown, with one line representing one gene across all fractions. Relative amounts of short and long 3’UTRs are represented by relative poly(A) site usage, with 50% signifying equal relative abundance of both isoforms. A 260 , UV absorbance of the gradient at 260nm (arbitrary unit). (E) Gene ontology analysis of genes for which short (blue) or long (red) 3’UTR isoforms are enriched in either translating or non-translating fractions from fly heads. The top four terms are shown for each category. Background set: all expressed genes.

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: Isolation, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) elav gene model (drawn to scale) and total RNA-seq tracks in fly heads of control and elav ΔnUTR flies. elav ΔnUTR flies are homozygous for an elav allele lacking the neuronal 3’UTR. (B) RT-qPCR quantification of the expression of the long elav 3’UTR isoform relative to total (short) elav mRNA in fly head polysome profile fractions, normalized to levels in tissue lysate. Error bars represent mean ±SD of two biological replicates. (C) ELAV protein expression in control and elav ΔnUTR adult fly heads. ELAV band intensity was normalized to the respective Histone H3 band intensity, with ELAV levels in control flies set to 1. Mean ±SD is indicated for three biological replicates. Ten heads of 3–5-day-old flies were used per replicate. (D) elav alleles used to create the fly genotypes used in ( E ). In elav FLAG , a Flag tag was inserted into the endogenous elav locus to generate a visibly larger protein. (E) ELAV protein expression by Western blot (top) and relative band intensity quantification (bottom) in adult fly heads of the indicated genotypes. Each genotype (all females) consists of two of the alleles shown in (D) . The proportion of ELAV expressed from each allele (control and elav FLAG , red; or elav ΔnUTR , light pink) was normalized to total ELAV per lane, with ELAV levels in elav FLAG /elav FLAG set to 1. Error bars represent mean ±SD of three biological replicates. *p ≤ 0.05 (Welch’s t-test). (F) Model of 3’UTR-mediated regulation of elav translation, in which mRNA isoforms containing the longer 3’UTR (nUTR, red) are translationally repressed and short isoforms produce the bulk of the encoded protein. ELAV protein in turn promotes the formation of longer 3’UTRs, negatively feeding back on ELAV abundance. (G, H) elav RNA ( G , males and females) and ELAV protein ( H , males) expression in adult heads of flies carrying increasing numbers of elav transgenes, to obtain the indicated gene dosage. Transgenes consisted of either the wild-type elav gene region (control) or lacked the elav nUTR ( ΔnUTR ). Genotypes are as follows, with + denoting a wild-type chromosome and tsg a chromosome carrying a transgene. Males, 1x (+/Y ; ; +/+ ) , 2x ( +/Y ; ; tsg/ +), 3x ( +/Y ; ; tsg/tsg ). Females, 0.5x ( Δelav/+ ; ; +/+) , 1x (+/+ ; ; +/+), 2x ( +/+; ; tsg/tsg ). For RT-qPCR, elav mRNA levels were normalized to RpL32 mRNA and levels in flies “control transgene, 1x dosage” were set to 1. For Western Blot, ELAV band intensity was normalized to the respective GAPDH band intensity, with ELAV levels in flies “1x dosage” set to 1. Error bars represent mean ±SD of three biological replicates for each genotype. Ten heads of adult flies were used per replicate.

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: RNA Sequencing, Control, Quantitative RT-PCR, Expressing, FLAG-tag, Western Blot

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Viability of elav ΔnUTR flies, measured as the rate of eclosion of live adult flies, compared to expected Mendelian ratios. Eclosion rates were normalized to those of control flies. Error bars indicate mean ±SD of four biological replicates. 375 (control) and 414 ( elav ΔnUTR ) adult flies were scored. **p ≤ 0.01 (Welch’s t-test). (B, C) Fraction of elav ΔnUTR and control flies that hatched from embryos into first instar larvae (B) , and that eclosed from pupae into live adults (C) , at the indicated time points after egg laying. Mean ±SEM of 10 biological replicates (10 embryos/pupae per replicate) is represented for each genotype. χ²=12.93, p=3.23 e-0.4 (B) , χ²=133.9, p=<0.0001 (C) (Mantel-Cox log-rank test). (D) Differential expression of the short (500 bp preceding the pPAS) and long 3′UTR in 313 ELAV target genes (genes that undergo ELAV-mediated 3′UTR extension), in elav ΔnUTR compared to control fly heads. Expression was quantified from RNA-seq data. ** p≤0.01 (Wilcoxon test). (E) Differential protein expression in elav ΔnUTR compared to control fly heads. Peptide intensity was measured by mass spectrometry. Proteins encoded by ELAV target genes are marked in red, with ELAV in black. (F) Expression of proteins encoded by ELAV target genes (top 50 genes with long 3’UTR isoform upregulated), in elav ΔnUTR compared to control fly heads. (G) RT-qPCR quantification of the long and short elav 3’UTR isoforms in adult heads of flies kept in control (with food) conditions or under starvation stress for 24h (starved). RNA levels were normalized to RpL32 mRNA and levels in control conditions were set to 1. Error bars represent mean ±SD of three biological replicates. (H, I) ELAV protein expression in adult heads of flies that were subjected to starvation stress for 24h. In (H), the Western blot membrane was cut below the ELAV band at 50 kDa and exposed separately to visualize degradation products. Intensities of the ELAV band and the ELAV degradation smear were normalized to the respective Histone H3 band intensity, with levels in control conditions set to 1. Mean ±SD is indicated for four (H) and three (I) biological replicates. (J) Stress resilience of control and elav ΔnUTR flies, measured as the proportion of flies alive after being subjected to starvation stress for the indicated number of hours. Mean ±SEM of five biological replicates (10 males and 10 females per replicate) is represented for each genotype. χ²=23.12, p=1.5 X10 −6 (Mantel-Cox log-rank test). (K) Principal component analysis of gene expression in adult heads of control and elav ΔnUTR flies kept in control (with food) conditions or under starvation stress for 24h (starved). (L) Differential expression of genes of the indicated gene sets in adult heads of flies kept under starvation stress for 24h compared to control fly heads. Expression was quantified from RNA-seq data. * p≤0.05 (Wilcoxon test).

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: Control, Quantitative Proteomics, Expressing, RNA Sequencing, Mass Spectrometry, Quantitative RT-PCR, Western Blot, Membrane, Gene Expression

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Flag xRIP-seq signal tracks for the elav 3′UTR, in flies in which Pumilio was endogenously Flag-tagged ( pum Flag ), and in untagged control flies (w 1118 ) . Signal is shown normalized to respective input. (B) ELAV protein expression in brains of control and Δpum larvae. Flies are homozygous for the elav FLAG allele on the first chromosome. (C) Western blot (top) and relative band intensity quantification (bottom) of ELAV protein in brains of control and Δpum larvae. All larvae are females expressing both Flag-ELAV (from the elav FLAG allele, red arrowhead) and untagged ELAV (from the elav ΔnUTR allele, light pink arrowhead). Histone H3 serves as a loading control. The proportion of ELAV expressed from each allele was normalized to total ELAV per lane, with ELAV levels in Control set to 1. Error bars represent mean ±SD of three biological replicates. *** p ≤ 0.001 (Welch’s t-test). (D) Enrichment of neuronal 3′UTRs in Pum xRIP relative to input. The heatmap profile plot displays 1 kb upstream, and the neuronal 3′UTR downstream (scaled region, indicated in red), of the proximal poly(A) site. Pum xRIP was performed in adult fly heads. (E) Quantification of Pum binding to short and nUTR-containing mRNA isoforms of ELAV target genes, by Pum xRIP-seq signal compared to input. p=0.0438 (Wilcoxon test). (F) Differential protein expression in Δpum compared to control fly heads. Peptide intensity was measured by mass spectrometry. Proteins encoded by Pum target genes (mRNAs enriched in Pum xRIP-seq compared to input) and proteins encoded by ELAV target genes (genes that undergo ELAV-mediated 3′UTR extension) are marked in purple and red, respectively. Pum is black. (G) Proportion and numbers of proteins encoded by transcripts of the indicated groups that are upregulated (up) and downregulated (down) in Δpum mutant larval brains compared to control. All mRNAs are compared to mRNAs whose long 3’UTR isoform is preferentially bound by Pum compared to the short isoform of the same gene. (H) Model of nUTR-mediated translational regulation of neuronal genes. nUTR-containing mRNAs are translationally repressed. Low levels of ELAV protein promotes expression of short 3’UTR isoforms, which in turn increases global protein abundance. The feedback loop is supported by nUTR-specific binding and translational repression by Pumilio.

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: Control, Expressing, Western Blot, Binding Assay, Mass Spectrometry, Mutagenesis, Quantitative Proteomics

Journal: bioRxiv

Article Title: Regulation of protein abundance in neurons by selective translation of 3′UTR isoforms

doi: 10.64898/2026.07.08.737200

Figure Lengend Snippet: (A) Gene model and mRNA-seq tracks of the ELAVL1 3’UTR (drawn to scale) in human iPSCs, and in the course of differentiation into neural progenitor cells and neurons. (B) Profile from NSC polysome profiling experiment. The four fractions used for downstream RNA-seq analysis are indicated. A 260 , UV absorbance of the gradient at 260nm (arbitrary unit). (C) Proportion of genes in NSCs that display differential translation of 3’UTR isoforms. Values indicate the number of genes for which isoform expression ratios are biased (compared to lysate) towards the short (blue) or long (red) 3’UTR isoform. (D) RT-qPCR quantification of the expression of the long ELAVL1 3’UTR isoform relative to total (short) elav mRNA isoforms in polysome profile fractions, normalized to levels in tissue lysate. Error bars represent mean ±SD of three biological replicates. (E) Light microscopy images of cerebral organoids grown from control and ELAVL1 ΔnUTR human iPSCs on day 14 and day 34. Scale bar: 500µm. (F) Mean diameter of control and ELAVL1 ΔnUTR (two independent mutants) cerebral organoids at the indicated number of days post-seeding. Error bars represent mean ±SD for six organoids per genotype and time point. (G) Apotome imaging of control and ELAVL1 ΔnUTR cerebral organoids stained for SOX2, ELAVL1 and DAPI. Scale bar: 400µm. (H) Quantification of neural rosette size in control and ELAVL1 ΔnUTR cerebral organoids. 36 (control), 31 (mutant #1) and 40 (mutant #2) rosettes were scored. ****p < 0.0001 (Mann-Whitney U test). (I) Quantification of the mean signal intensity for SOX2 (background intensity-corrected) in control and ELAVL1 ΔnUTR organoids. Error bars represent mean ±SD for four replicates. ****p < 0.0001 (Mann-Whitney U test). (J) Western blot comparing ELAVL1 protein expression in control and ELAVL1 ΔnUTR day 5 organoids. ELAVL1 band intensities were normalized to the respective Histone H3 band intensity, with ELAVL1 levels in control organoids set to 1. Mean ±SD is indicated for three biological replicates. (K) Quantification of the ELAVL1/SOX2 signal intensity ratio in control and ELAVL1 ΔnUTR organoids. Error bars represent mean ±SD for four replicates. *p < 0.05 (Welch’s t-test).

Article Snippet: Cells were transfected with RNP complexes and StemMACSTM eGFP mRNA (Miltenyi Biotec, 130-101-114).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Light Microscopy, Control, Imaging, Staining, Mutagenesis, MANN-WHITNEY, Western Blot

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Schematic illustration of in vivo tumor immunotherapy enhanced by mRNA/HNPs through intravenous injection. H18 lipid, DOPE, cholesterol, DMG-PEG 2000 and mRNA were mixed to form mRNA/H 18 NPs with the special multilamellar concentric nanostructures. Following intravenous administration, mRNA/H 18 NPs demonstrated preferential adsorption of complement C3 proteins to form a characteristic protein corona, resulting in specific mRNA transfection in the spleen, especially in splenic dendritic cells. When encapsulating tumor antigen-encoding mRNA, the mRNA/H 18 NPs achieved precise transfection of the antigen mRNA in splenic dendritic cells. This targeted delivery stimulated dendritic cell maturation and subsequent antigen presentation, initiating robust T cell priming. The activated antigen-specific cytotoxic T lymphocytes then infiltrated into tumor tissues, ultimately inducing tumor cell elimination.

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: In Vivo, Injection, Adsorption, Transfection, Immunopeptidomics

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Preparation and Characterization of Optimal mRNA/H 18 NPs. (A) Schematic illustration of mRNA/H 18 NPs preparation. (B) The size distribution and (C) zeta potential of optimized mRNA/H 18 NPs. (D) The apparent p K a of mRNA/H 18 NPs. (E) Cryo-EM image of optimized mRNA/H 18 NPs. Scale bar = 50 nm. (F) Representative image and percentage of bioluminescence in major organs of mice following intravenous injection of mLuc/H 18 NPs. (G)-(I) Stability test for mRNA/H 18 NPs. (G) Size and PDI of mRNA/H 18 NPs when stored at 4 °C for different days (0, 3, 5, 7). (H) Left: Total bioluminescence flux in the spleen of mice 6 h after intravenous injection of mLuc/H 18 NPs stored at 4 °C for different days (0, 3, 5, 7). Right: Representative bioluminescence images of major organs of mice 6 h after intravenous injection of different mLuc/H 18 NPs stored at 4 °C for different days (0, 3, 5, 7). (I) Size and PDI of mRNA/H 18 NPs when diluted with PBS by different times. Data were shown as mean ± SD (n = 3).

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: Zeta Potential Analyzer, Cryo-EM Sample Prep, Injection

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: In vivo splenic DC-specific transfection of mRNA/H 18 NPs and in vitro protein corona analysis of mRNA/H 18 NPs. (A) EGFP protein expression in splenic cell subsets of C57BL/6J mice 24 h post intravenous injection of different formulations. (B) The top 5 most abundant plasma proteins adsorbed on mRNA/H 18 NPs (C3: Complement C3; Ighm: Immunoglobulin heavy constant mu; Hbat1: Alpha-globin; Itih4: Inter alpha-trypsin inhibitor, heavy chain 4; Cnn2: Calponin). (C) Heatmap plot of major proteins in the protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. PBS group was used as a negative control. (D) Quantification of major adsorbed protein categories of different formulations. (E) Complement C3 abundance in protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. (F) Bioluminescence images of major organs and (G) Quantification of total bioluminescence flux in the spleen from C57BL/6J mice 6 h after intravenous injection of mLuc/H 18 NPs (mLuc dose of 0.25 mg kg −1 ). Mice were pre-treated with cobra venom factor (CVF) or PBS. (H) Fluorescence quantification of Cy5 mRNA delivered by uncoated or complement C3-coated Cy5-mRNA/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. (I) Bioluminescence intensity of luciferase protein translated from mRNA delivered by uncoated or complement C3-coated mLuc/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. Data were shown as mean ± SD (n = 3).

Article Snippet: Luciferase mRNA, OVA mRNA, and EGFP mRNA were purchased from Novoprotein (Suzhou, China).

Techniques: In Vivo, Transfection, In Vitro, Expressing, Injection, Clinical Proteomics, Negative Control, Combined Bisulfite Restriction Analysis Assay, Fluorescence, Incubation, Blocking Assay, Luciferase

Journal: bioRxiv

Article Title: An Engineered Halotolerant Chimeric T7 RNA Polymerase for High-Yield, Low-Immunogenicity Synthesis of RNA via Simple Batch Transcription

doi: 10.64898/2026.05.13.724829

Figure Lengend Snippet: The DNA-binding domain (DBD)-fused mutants (T7 M4/M5/M6; Sso7d/MC1), utilized at a 50% reduced dosage, demonstrated superior RNA titers compared to the wild-type enzyme. Under 100–150 mM NaCl, these chimeric polymerases significantly mitigated dsRNA byproduct formation and enhanced 3’ homogeneity. This optimized catalytic profile facilitates high-efficiency, “one-pot” synthesis of both conventional mRNA and circRNA.

Article Snippet: Concurrently, DNA templates for the IVT of PiggyBac (PB) mRNA, eGFP mRNA and eGFP circular RNA (circRNA)—all driven by a T7 promoter—were synthesized by General Biosystems.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: An Engineered Halotolerant Chimeric T7 RNA Polymerase for High-Yield, Low-Immunogenicity Synthesis of RNA via Simple Batch Transcription

doi: 10.64898/2026.05.13.724829

Figure Lengend Snippet: (A) Schematic of the experimental workflow: transcripts of varying architecture and length— including linear eGFP mRNA (∼1 kb), PB mRNA (2.1 kb), and circular eGFP RNA (circRNA, ∼1.7 kb) - were synthesized utilizing the specified RNAP mutants at 0, 100, or 150 mM NaCl. These preparations were transfected into HEK293T and THP-1 cell lines to evaluate translational efficiency and innate immune activation (via IFN-β secretion). (B, E) Mean fluorescence intensity (MFI) of eGFP expression in HEK293T cells at 48 and 96 h post-transfection for linear mRNA (B) and circRNA (E). (C, F) IFN-β secretory profiles of THP-1 cells following stimulation with linear mRNA (C) and circRNA (F) preparations. (D, G) Quantitative assessment of residual dsRNA content within linear mRNA (D) and circRNA (G) cohorts. (H) Longitudinal eGFP expression over 288 h; upper panel summarizes four circRNA mutants with distinct splicing introns, lower panels show individual trajectories. Notably, DBD-tethered chimeras maintained robust translational potency and negligible immunogenic signatures across all ionic strengths. Conversely, wild-type (WT) T7 and the T7(G47A/P884G) mutant exhibited a marked attenuation in transcriptional performance and product bioactivity under elevated salt conditions. Data are expressed as mean ±s.d. (n=3).

Article Snippet: Concurrently, DNA templates for the IVT of PiggyBac (PB) mRNA, eGFP mRNA and eGFP circular RNA (circRNA)—all driven by a T7 promoter—were synthesized by General Biosystems.

Techniques: Synthesized, Transfection, Activation Assay, Fluorescence, Expressing, Mutagenesis

Journal: Journal of Nanobiotechnology

Article Title: Mechanistic insights into mRNA–LNP interactions: role of ionizable lipid content in regulating mRNA intracellular release and translation

doi: 10.1186/s12951-026-04383-5

Figure Lengend Snippet: Transfection efficiency of mRNA-LNPs. ( a ) EGFP expression in HEK293 cells transfected with EGFP-mRNA-LNPs (Moderna and Pfizer/BioNTech formulations). Expression was quantified by flow cytometry and presented as mean fluorescence intensity (MFI). ( b ) Luciferase expression following transfection with Fluc-mRNA-LNPs. ( c ) OVA expression following transfection with OVA-mRNA-LNPs (Moderna formulation). OVA expression was assessed by ELISA. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: Fig. 2 Transfection efficiency of mRNA-LNPs. ( a ) EGFP expression in HEK293 cells transfected with EGFP-mRNA-LNPs (Moderna and Pfizer/BioNTech formulations).

Techniques: Transfection, Expressing, Flow Cytometry, Fluorescence, Luciferase, Formulation, Enzyme-linked Immunosorbent Assay

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Poking Pluripotency: Nanoinjection Into Human iPSCs

doi: 10.1002/adma.202521046

Figure Lengend Snippet: — Workflow of the NT‐mediated cargo delivery of mRNA to hiPSCs. Briefly, hiPSCs were interfaced with mRNA‐loaded NTs and analyzed the next day for the expression of a reporter protein (mCherry, GFP, YPet). a‐i) Surface activation with UV/ozone. a‐ii) Surface functionalization with poly‐D‐lysine (PDL, mol. wt.: 1–5 kDa). a‐iii) NT loading with mRNA (85 ng/µL). a‐iv) Removal of the cargo supernatant. b‐i) ROCK inhibitor preconditioning of the hiPSCs with 20 µM Y‐27632. b‐ii) Dissociation of the hiPSC colonies using Accutase. c‐i) hiPSC seeding onto the NT array (single cells). c‐ii) Centrifugation of cells onto NTs (220 g, 15 min). c‐iii) Cargo uptake from mRNA‐loaded NTs. c‐iv) In situ extracellular matrix (ECM) coating (Matrigel). c‐v) Analysis of expression using confocal microscopy imaging and flow cytometry (reporter proteins). Figure partly created with biorender.com.

Article Snippet: Notably, without drying of the PDL before rinsing, transfection was less consistent (Figure ). mRNA was diluted to about 85 ng/μL using UP DI, pipetted onto the chips (4.5 μL/chip, held on the chip by surface tension), kept on ice (to maintain mRNA integrity and minimize evaporation), and placed on an orbital shaker (45 rpm), while samples were incubated for 1 h. Used mRNA was: mCherry‐encoding mRNA (#L‐7203, TriLink, CleanCap, 5moU, 1 μg/μL), eGFP‐encoding mRNA (#L‐7601, TriLink, CleanCap, 5mU, 1 μg/μL), YPet‐encoding mRNA (#130‐120‐971, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL), mCherry‐encoding mRNA (Messenger Bio, Cap1, N1‐methylpseudouridine, 1 μg/μL), and eGFP‐encoding mRNA (#130‐101‐114, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL).

Techniques: Expressing, Activation Assay, Centrifugation, In Situ, Confocal Microscopy, Imaging, Flow Cytometry

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Poking Pluripotency: Nanoinjection Into Human iPSCs

doi: 10.1002/adma.202521046

Figure Lengend Snippet: — NT array chip design, NT geometry, and cargo loading. a) Silicon wafer piece with 4×4 patterns, as used for DRIE processing. Inset: 4×4 mm 2 wafer piece containing only NTs, as used for transfection experiments. b) Scanning electron microscopy (SEM) images of the NT arrays (array arrangement and NT dimensions). The array pitch was 3 µm, and the NT length was about 3.2 µm (large‐scale overview in Figure ). c) Close up of the NT tip. The NT diameter was about 1 µm, and the rim thickness at the tip was less than 50 nm. d) Confocal microscopy images (top‐view) of NTs loaded with fluorescently‐labeled mRNA (Cy5‐tagged mRNA). The NT pattern was visible in the overview (dots), and steady Cy5 signal from the NTs illustrated homogeneous loading across the NT array (brightness quantification in Figure ).

Article Snippet: Notably, without drying of the PDL before rinsing, transfection was less consistent (Figure ). mRNA was diluted to about 85 ng/μL using UP DI, pipetted onto the chips (4.5 μL/chip, held on the chip by surface tension), kept on ice (to maintain mRNA integrity and minimize evaporation), and placed on an orbital shaker (45 rpm), while samples were incubated for 1 h. Used mRNA was: mCherry‐encoding mRNA (#L‐7203, TriLink, CleanCap, 5moU, 1 μg/μL), eGFP‐encoding mRNA (#L‐7601, TriLink, CleanCap, 5mU, 1 μg/μL), YPet‐encoding mRNA (#130‐120‐971, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL), mCherry‐encoding mRNA (Messenger Bio, Cap1, N1‐methylpseudouridine, 1 μg/μL), and eGFP‐encoding mRNA (#130‐101‐114, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL).

Techniques: Transfection, Electron Microscopy, Confocal Microscopy, Labeling

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Poking Pluripotency: Nanoinjection Into Human iPSCs

doi: 10.1002/adma.202521046

Figure Lengend Snippet: — hiPSCs interfaced with NT arrays imaged with SEM and confocal microscopy. a) Overview SEM image of hiPSCs cultured on the NT arrays (additional examples in Figure ). b) Close‐up view of an individual cell (magnified from panel a). c) Viability staining of hiPSCs cultured overnight on NTs (viable cells stained with Calcein (Ca), green; dead cells stained with propidium iodide (PI), red). Hoechst 33342 nuclear counterstain and flat controls shown in Figure . d) Cell viability of hiPSCs cultured on flat/blank, flat/mRNA, and NTs/mRNA derived from the PI channel (Ca‐derived cell viability in Figure ). e) Confocal microscopy images of hiPSCs on mCherry‐mRNA‐loaded NTs before harvesting and while expressing mCherry, red (cells stained with Ca, green/gray). Inset: The NT array was visible by dark spot pattern in the cytosol, indicating indentations of the cell membrane (focal plane at the NT tips). mCherry was imaged with the focal plane at the cell center (further away from the NTs). Additional examples in Figure . ANOVA with post hoc Tukey's test, n.s.: non‐significant, n = 3.

Article Snippet: Notably, without drying of the PDL before rinsing, transfection was less consistent (Figure ). mRNA was diluted to about 85 ng/μL using UP DI, pipetted onto the chips (4.5 μL/chip, held on the chip by surface tension), kept on ice (to maintain mRNA integrity and minimize evaporation), and placed on an orbital shaker (45 rpm), while samples were incubated for 1 h. Used mRNA was: mCherry‐encoding mRNA (#L‐7203, TriLink, CleanCap, 5moU, 1 μg/μL), eGFP‐encoding mRNA (#L‐7601, TriLink, CleanCap, 5mU, 1 μg/μL), YPet‐encoding mRNA (#130‐120‐971, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL), mCherry‐encoding mRNA (Messenger Bio, Cap1, N1‐methylpseudouridine, 1 μg/μL), and eGFP‐encoding mRNA (#130‐101‐114, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL).

Techniques: Confocal Microscopy, Cell Culture, Staining, Derivative Assay, Expressing, Membrane

Journal: Advanced Materials (Deerfield Beach, Fla.)

Article Title: Poking Pluripotency: Nanoinjection Into Human iPSCs

doi: 10.1002/adma.202521046

Figure Lengend Snippet: — Nanoinjection of various mRNA constructs into hiPSCs, including co‐transfection. Quantification of transfection yield via flow cytometry analysis and cell visualization via confocal microscopy. a) Exemplary flow cytometry plots (histograms of mCherry intensity) of hiPSCs harvested from blank, i.e., non‐loaded, flat silicon chips (flat/blank, negative control, light gray), cargo‐loaded flat silicon chips (flat/mRNA, topography control, gray), and cargo‐loaded NT arrays (NTs/mRNA, red), cargo: mCherry‐expressing mRNA. b) Proportion of mCherry‐positive cells harvested from flat/blank, flat/mNRA, and NTs/mRNA, cargo: mCherry‐expressing mRNA. Quantification via flow cytometry; mCherry intensity gated to <1% of cells harvested from flat/blank silicon ( n = 8, entire gating stream including back‐gating in Figure a). c) Exemplary intensity plots of hiPSCs nanoinjected with GFP and YPet mRNA. d) Transfection yields using GFP and YPet mRNA, n = 3–5. e) Exemplary confocal microscopy images of hiPSCs harvested from NT arrays after NT‐transfection with mCherry/GFP/YPet‐expressing mRNA, and replated onto Matrigel‐coated imaging dishes (10 µM Y‐27632 ROCK inhibitor, ∼4 h after seeding). The reporter‐positive hiPSCs showed homogeneous distribution of reporter fluorescence inside the cytosol with varying intensities between different cells (nuclear counterstain: Hoechst 33342). f) Exemplary mCherry/GFP intensity plot of co‐nanoinjected hiPSCs. g) Quantification of double‐positive hiPSCs. n = 5. h) Quantification of Pearson's correlation coefficient of double‐positive hiPSCs. i) Exemplary confocal images of reseeded co‐nanoinjected hiPSCs, including merge of mCherry and GFP channel (additional examples in Figure ). All intensity histograms used for quantification are compiled in Figure . ANOVA with post hoc Tukey's test, n.s.: non‐significant, *** p < 0.001, n = 3–8.

Article Snippet: Notably, without drying of the PDL before rinsing, transfection was less consistent (Figure ). mRNA was diluted to about 85 ng/μL using UP DI, pipetted onto the chips (4.5 μL/chip, held on the chip by surface tension), kept on ice (to maintain mRNA integrity and minimize evaporation), and placed on an orbital shaker (45 rpm), while samples were incubated for 1 h. Used mRNA was: mCherry‐encoding mRNA (#L‐7203, TriLink, CleanCap, 5moU, 1 μg/μL), eGFP‐encoding mRNA (#L‐7601, TriLink, CleanCap, 5mU, 1 μg/μL), YPet‐encoding mRNA (#130‐120‐971, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL), mCherry‐encoding mRNA (Messenger Bio, Cap1, N1‐methylpseudouridine, 1 μg/μL), and eGFP‐encoding mRNA (#130‐101‐114, Miltenyi Biotech, capped, pseudouridine and 5‐methyl‐cytidine, lyophilized: 20 μg, reconstituted: 500 ng/μL).

Techniques: Construct, Cotransfection, Transfection, Flow Cytometry, Confocal Microscopy, Negative Control, Control, Expressing, Imaging, Fluorescence