human lin28a expression  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

doi: 10.1093/nar/gkx298

Figure Lengend Snippet: RNA delivery of LIN28A -coding mRNA and LIN28A-responsive devices. ( A ) Schematic representation of RNA delivery of LIN28A -coding mRNA, LIN28A-responsive mRNA and reference mRNA. 293FT cells were co-transfected with three synthetic mRNAs, trigger mRNA ( LIN28A or CBG68Luc mRNA), LIN28A-responsive hmAG1 mRNA and iRFP670 mRNA (reference mRNA) as a transfection control. ( B ) Translational efficiencies of LIN28A-responsive mRNAs. Transfection was performed in a 24-well format. Error bars represent mean ± SD ( n = 3 independent experiments, each performed in triplicate). * P < 0.05 (Welch's t -test).

Article Snippet: A stable human cell line expressing streptavidin binding protein (SBP)-fused lin28A (SBP-lin28A) was established by Flp Recombinase-Mediated Integration (Flp-In system, Invitrogen) according to the manufacturer's instructions.

Techniques: Transfection, Control

Journal: Nucleic Acids Research

Article Title: Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

doi: 10.1093/nar/gkx298

Figure Lengend Snippet: Distinction of living cell types using LIN28A-responsive mRNA devices. ( A ) Distinction of human iPSCs with differentiated cells by detecting endogenous LIN28A expression level. LIN28A is highly expressed in iPSCs, and its expression level is decreased after differentiation. LIN28A in iPSCs reduces the translation level from LIN28A-responsive hmAG1 mRNA. In differentiated cells, LIN28A expression level is low, resulting in normal translation (left and middle panels). After co-transfection of the LIN28A-responsive hmAG1 mRNA and reference mRNA encoding mKO2, the cells typically show belt-like distribution in a two-dimensional plot by flow cytometry analysis (right panel). A shift of the population is observed as the result of translational repression of hmAG1 in iPSCs but not in differentiated cells, resulting in the separation of iPSCs from differentiated cells based on LIN28A detection. ( B ) Relative translational efficiency of transfected mRNAs in iPSCs and D14. Relative translational efficiency = (N.I. of iPSCs)/(N.I. of D14) (See ‘Flow cytometry data analysis’ in ‘Materials and Methods’ section). Error bars represent mean ± SD ( n = 3 independent experiments, each performed in duplicate). *** P < 0.0005 (Welch's t -test). ( C ) Representative overlaid dot plots of iPSCs (red) and differentiated cells (D14, blue). ( D ) Representative histograms related to Figure . Red, hiPSCs; blue, D14. ( E ) Western blot analysis of the LIN28A expression. A total of 0 ng Dox, 10 ng Dox and 293FT were applied as controls, and GAPDH was used as internal control. The samples used in these experiments were also used for the data in Figure . iPSCs and D14 samples were collected in different experiments. LIN28A was detected by anti-LIN28A. ( F ) Endogenous miRNA expression levels by LIN28A-responsive mRNA transfection. Mature let7d, let7g and miR-98 levels were analyzed by qRT-PCR. We used differentiated cells (D14, black bar) as a control, because the expression levels of these miRNAs increased in differentiated cells compared with iPSCs. Thus, the expression levels of miRNAs in untransfected (white), mRNA (without aptamer)-transfected (blue) and stbC-containing mRNA-transfected (red) iPSCs were normalized by the expression level of the miRNAs in D14. Error bars indicate SD for three independent experiments. Significant differences were not observed among untransfected, no aptamer and stbC.

Article Snippet: A stable human cell line expressing streptavidin binding protein (SBP)-fused lin28A (SBP-lin28A) was established by Flp Recombinase-Mediated Integration (Flp-In system, Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Cotransfection, Flow Cytometry, Transfection, Western Blot, Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

doi: 10.1093/nar/gkx298

Figure Lengend Snippet: Construction of LIN28A-responsive devices. ( A ) Predicted secondary structures of the LIN28A aptamers used in this study. Small letters represent the nucleotides changed from the original preE-let7d sequence. Purple shadows represent the LIN28A recognition sequences. Color scale represents base pairing probabilities calculated by CentroidFold software. ( B ) Overlaid dot plots of the flow cytometry analysis. Red, cells co-transfected with EGFP reporter plasmid and trigger plasmid encoding LIN28A and tagRFP. Blue, cells co-transfected with EGFP reporter plasmid and control plasmid without LIN28A. ( C ) Translational efficiency of LIN28A-responsive devices. All values were normalized by cells transfected with a plasmid lacking aptamers (No aptamer). Transfection was performed in a 24-well format. Error bars represent mean ± SD ( n = 3 independent experiments, each performed in triplicate). ( D ) Translational efficiency of defective LIN28A-responsive devices (sequences are described in ). All values were normalized by cells transfected with No aptamer. Transfection was performed in a 96-well format. ( E ) Translational efficiency using mutant LIN28A (W46A, LIN28A mut ), which weakens RNA binding. All values were normalized by cells co-transfected with No aptamer and trigger plasmid coding for wild-type LIN28A. Transfection was performed in a 24-well format. Error bars represent mean ± SD ( n = 3 independent experiments, each performed in triplicate). ( F ) Translational efficiency of LIN28A-responsive devices in HeLa cells. Transfection was performed in a 24-well format. Error bars represent mean ± SD ( n = 3 independent experiments, each performed in triplicate). All values were normalized as described in D. * P < 0.05, ** P < 0.005, *** P < 0.0005 (Welch's t -test).

Article Snippet: A stable human cell line expressing streptavidin binding protein (SBP)-fused lin28A (SBP-lin28A) was established by Flp Recombinase-Mediated Integration (Flp-In system, Invitrogen) according to the manufacturer's instructions.

Techniques: Sequencing, Software, Flow Cytometry, Transfection, Plasmid Preparation, Control, Mutagenesis, RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells

doi: 10.1093/nar/gkx298

Figure Lengend Snippet: Quantitative detection of LIN28A expression by mRNA devices. ( A ) Expected behavior of the LIN28A device in response to LIN28A protein expression. LIN28A is generated in a Dox-dependent manner, and the translation efficiency of the device relies on the expression level of intracellular LIN28A. At low Dox concentration, the expression level of LIN28A is also low and the device does not repress EGFP translation (left side). At high Dox concentration, LIN28A expression is also high and the intensity of EGFP decreases because of translational repression (right side). ( B ) Relationship between relative fluorescence intensity (translation of mRNA) and Dox concentration. Relative intensities = (N.I.)/(N.I. without Dox induction). (The relative intensities were normalized to the intensity at 0 ng Dox. See ‘Flow cytometry data analysis’ in ‘Materials and Methods’ section.) Error bars are mean ± SD. The statistical analysis was performed between No aptamer and stbC. ** P < 0.005 (Welch's t -test). ( C ) Western blot analysis of LIN28A expression at 24 h after transfection. Tag sequence-fused LIN28A was induced by Dox. LIN28A was detected by anti-LIN28A. GAPDH was used as internal control. The concentration of Dox ranged between 0 and 10 ng/ml. Relationship between relative LIN28A amount calculated from the western blotting analysis in Figure and Dox concentration. Reporter expression from the device was controlled by the Dox concentration. Error bars are mean ± SD. ( E ) Relationship between the relative intensity of EGFP produced from the device and LIN28A amount. Plotted data are same as in Figure and . The maximum value of LIN28A/GAPDH in Figure was taken from the minimum value of LIN28A/GAPDH between 3 and 10 ng/ml of Dox condition in Figure . Blue, No aptamer; orange, preE-let7d; red, stbC. R 2 , coefficient of determination.

Article Snippet: A stable human cell line expressing streptavidin binding protein (SBP)-fused lin28A (SBP-lin28A) was established by Flp Recombinase-Mediated Integration (Flp-In system, Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Generated, Concentration Assay, Fluorescence, Flow Cytometry, Western Blot, Transfection, Sequencing, Control, Produced