glypican 1 gfp pcdna3 1  (Addgene inc)

Journal: bioRxiv

Article Title: 3’ to 5’ translation of linear mRNAs?

doi: 10.64898/2025.12.29.696803

Figure Lengend Snippet: (A) Diagram showing pcDNA3.1(+) ZKSCAN1 MCS as the vector for circular mRNA backward translation. The construct consisted of two backward eGFP fragments (coordinates: 593→1 and 720→594), separated by a TIS-containing sequence. The TIS was flanked by a backward start codon (←GUA) and a backward stop codon (AAU←). (B) Representative fluorescence microscopy images illustrating positive cells after transfection of HEK-293T cells with circular (bw)-eGFP (w/o TIS), hs .circIFITM1-TIS-(bw)-eGFP, hs .circCAPN15-TIS-(bw)-eGFP, and hs .circSCRIB-TIS-(bw)-eGFP. Scale bar, 20μm. Live cells were stained with Mito-Tracker (red, mitochondria). (C) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the empty vector plasmid, as the negative control. Scale bar, 60μm. HEK-293T cells transfected with the Addgene plasmid #69909 (pcDNA3.1(+) ZKSCAN1 MCS-circ (fw)-eGFP) served as the positive control, with approximately 35%–40% of cells displayed green fluorescence. Scale bar, 40μm. (D) Diagram showing pcDNA3.1(+) as the vector for linear mRNA backward translation. A backward GUA (←GUA) and a backward stop codon (AAU←) were inserted before the TIS. (E) Representative fluorescence microscopy images illustrating positive cells after transfection of HEK-293T cells with linear (bw)-eGFP (w/o TIS), hs .circIFITM1-TIS-(bw)-eGFP, hs .circCAPN15-TIS-(bw)-eGFP, and hs .circSCRIB-TIS-(bw)-eGFP. Scale bar, 20μm. Live cells were stained with Mito-Tracker (red, mitochondria). (F) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the empty vector plasmid, as the negative control. Scale bar, 60μm. HEK-293T cells transfected with the pcDNA3.1-linear (fw) -eGFP plasmid as the positive control with approximately 85–90% of cells displayed green fluorescence. Scale bar, 60μm. (G) Comparison of linear and circular backward translation effiency in the HEK-293T transfection experiments. The Y axis shows the ratio of positive cells after transfection of HEK-293T cells with circular RNA and linear RNA, respectively. Statistical comparisons between groups were performed using Mann-Whitney U in GraphPad Prism (version 9.2.0). Statistical significance: ns: not significant, “*”: p <0.05, “**”: p <0.01, “***”: p <0.001, “****”: p <0.0001.

Article Snippet: The vector pcDNA3.1(+) ZKSCAN1 MCS + Sense IRES (Addgene: #69909) was linearized using HindIII and XbaI.

Techniques: Plasmid Preparation, Construct, Sequencing, Fluorescence, Microscopy, Transfection, Staining, Negative Control, Positive Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: 3’ to 5’ translation of linear mRNAs?

doi: 10.64898/2025.12.29.696803

Figure Lengend Snippet: (A) Diagram showing design of the pcDNA3.1(+) vector for bidirectional translation of linear mRNAs. The backward (bw)-eGFP was placed upstream of the TIS, and the forward (fw) mScarlet3 was placed downstream of the TIS. (B) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed linear bidirectional fluorescence (bw)-eGFP- hs .circIFITM1-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (C) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed linear bidirectional fluorescence (bw)-eGFP- dm .circSCRIB-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (D) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the linear constructed bidirectional fluorescence (bw)-eGFP- hs .circCAPN15-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (E) Diagram showing design of the pcDNA3.1(+) ZKSCAN1 MCS vector for bidirectional translation of circular RNAs. The backward (bw)-eGFP was placed upstream of the TIS, and the forward (fw) mScarlet3 was downstream of the TIS. (F) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent circular (bw)-eGFP- dm .circSCRIB-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (G) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent (bw)-eGFP- hs .circCAPN15-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (H) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent (bw)-eGFP- hs .circIFITM1-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (I) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent (bw)-mScarlet3- hs .circIFITM1-TIS-(fw)-eGFP. Scale bar, 20 µm. Quantifications of the fluorescence positive cells ratio after transfection were shown below each microscopy images panel.

Article Snippet: The vector pcDNA3.1(+) ZKSCAN1 MCS + Sense IRES (Addgene: #69909) was linearized using HindIII and XbaI.

Techniques: Plasmid Preparation, Fluorescence, Microscopy, Transfection, Construct

Journal: Nucleic Acids Research

Article Title: Internal ribosome entry sites enhance translation in trans in antisense non-coding SINEUP and circular RNAs

doi: 10.1093/nar/gkaf788

Figure Lengend Snippet: The invSINEB2 sequence shows IRES activity in cis . ( A ) Schematic representation of pRUF dual-luciferase reporter vectors. The negative control, empty pRUF-MCS (NO IRES), contains the CDS of both Rluc (pale green) and Fluc (pale yellow), with an MCS in between. Rluc is constitutively expressed and translated in a cap-dependent manner, whereas Fluc expression depends on the presence of an upstream candidate sequence driving translation. The pRUF-c-myc IRES (c-myc IRES, dark gray) vector was used as a positive control. The AS Uchl1 invSINEB2 (light gray) and dirSINEB2 (gray) elements were tested as candidate sequences. ( B ) IRES activity (bicistronic plasmids). Luciferase activity was measured 48 h after transfection. IRES activity was calculated as the ratio of Fluc to Rluc (Fluc/Rluc) luminescence and normalized to the negative control (NO IRES). Results show that the invSINEB2 element induced Fluc protein translation in cis . Plots represent the IRES activity (mean ± SD) from independent biological replicates ( n = 5) performed in duplicate. Statistical significance was determined using ordinary one-way ANOVA with Holm–Sidak's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Comparisons were made against the control condition unless otherwise specified. ( C ) Fluc/Rluc mRNA ratio (bicistronic plasmids). The Fluc/Rluc mRNA ratio was also calculated for each sample to confirm the absence of cryptic promoter activity, with no significant variation observed. ( D ) Validation of bicistronic transcript integrity. To confirm the absence of splicing events mediated by the inv/dirSINEB2-spacer sequences, cDNA was amplified using two different PCRs (PCR1 and PCR2). In PCR1, a pSV40 forward primer (annealing downstream of the transcription start site and upstream of the Rluc CDS, light gray arrows) was used in combination with a reverse primer targeting the Rluc CDS (gray arrows). PCR2 was performed using the same pSV40 forward primer and a reverse primer annealing to the Fluc CDS (pale yellow arrows). The resulting amplicons were visualized on an agarose gel. Double bands represent long and short amplicons derived from unprocessed and processed transcripts, respectively, due to an intronic sequence downstream of the pSV40 promoter in the pRUF vector backbone. All amplicons showed the expected sizes, confirming the transcription of a single “bicistronic” RNA for each construct. PCR2 further confirmed a single full-length transcript of ∼2, 2.4, and 2.3 kb for the NO IRES, c- myc IRES, and inv-dirSINEB2 constructs, respectively, demonstrating the absence of splicing events mediated by the inv/dirSINEB2-spacer sequences. ( E ) IRES activity (IVT mRNA). Bicistronic mRNAs were in vitro transcribed and transfected into HEK293T cells. NO IRES and c- myc IRES were used as negative and positive controls, respectively. Luciferase activity was measured 6 h post-transfection. IRES activity was represented as the Fluc/Rluc activity ratio and calculated relative to the negative control (NO IRES). Data show that the invSINEB2 element induced Fluc protein translation in cis . Plots represent the IRES activity (mean ± SD) from independent biological replicates ( n = 4) performed in duplicate. Statistical significance was determined using ordinary one-way ANOVA with Holm–Sidak's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Comparisons were made against the control condition unless otherwise specified. ( F ) Schematic representation of circRNA reporter vectors. The pCDNA3.1(+) ZKSCAN1 MCS-WT Split GFP + SENSE vector consists of two complementary introns flanking two GFP exon fragments (in reversed order). The MCS is placed between the two reversed GFP exon fragments, allowing the evaluation of whether a sequence of interest can drive the formation of a circRNA encoding GFP protein upon backsplicing, thereby testing its ability to support cap-independent GFP translation. A circGFP RNA construct containing an IRES in an inverted orientation between the two GFP-coding fragments was used as a negative control. The c- myc IRES (dark gray) and invSINEB2 elements were cloned into the MCS. The c- myc IRES vector served as a positive control for cap-independent protein translation. ( G ) IRES activity (circGFP RNA). circGFP RNAs were transfected into HEK293T cells. circGFP RNA with an inverted IRES between the two GFP-coding fragments (NO IRES) and circGFP RNA containing the c- myc IRES were used as negative and positive controls, respectively. Fluorescence was measured 48 h post-transfection using a flow cytometer. Data show that the invSINEB2 element induced GFP protein translation in a cap-independent manner. Results are presented as the fluorescence percentage. Plots represent the IRES activity (mean ± SD) from independent biological replicates ( n = 5) performed in duplicate. Statistical significance was determined using ordinary one-way ANOVA with Holm–Sidak's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Comparisons were made against the control condition unless otherwise specified. ( H ) RT–qPCR data corresponding to (G). Circular (left) and linear (right) RNA expression was detected via RT–qPCR. Plots represent the fold change in RNA expression levels (mean ± SD) from independent biological replicates ( n = 3). Statistical significance was determined using ordinary one-way ANOVA with Holm–Sidak's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Comparisons were made against the control condition.

Article Snippet: To generate constructs for the circular GFP reporter assay, the pcDNA3.1(+) ZKSCAN1 MCS-WT Split GFP+ Sense IRES vector (Addgene, Plasmid #69 909) was used.

Techniques: Sequencing, Activity Assay, Luciferase, Negative Control, Expressing, Plasmid Preparation, Positive Control, Transfection, Control, Biomarker Discovery, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Construct, In Vitro, Clone Assay, Fluorescence, Flow Cytometry, Quantitative RT-PCR, RNA Expression