Journal: PLoS ONE

Article Title: In Vivo Cell Reprogramming towards Pluripotency by Virus-Free Overexpression of Defined Factors

doi: 10.1371/journal.pone.0054754

Figure Lengend Snippet: Balb/C mice were HTV injected with 0.9% saline alone, 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline and at days 2, 4, 8, 12, 24, RT-qPCR analysis of hepatocytes was performed to determine the relative gene expression of: ( a ) transfected transcription factors (OKSM) and ( b ) endogenous pluripotency markers. All gene expression levels were normalized to HTV-injected saline group (*p<0.05 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison); ( c ) flow cytometry analysis of OCT3/4 positive and NANOG positive cells in liver extracts; ( d ) relative gene expression of hepatocyte markers as determined by RT-qPCR. All gene expression levels were normalized to saline HTV-injected group (* p<0.05 indicates statistically significant differences between the expression levels for hepatocyte markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison).

Article Snippet: Reprogramming plasmids pCX-OKS-2A encoding OCT3/4, KLF4, SOX2 ; pCX-cMyc encoding CMYC and pCAG-GFP encoding eGFP under the control of CAG promoter (as previously described by Okita et al. ) were obtained from Addgene (USA) as bacterial stabs.

Techniques: Injection, Quantitative RT-PCR, Expressing, Transfection, Flow Cytometry

Journal: PLoS ONE

Article Title: In Vivo Cell Reprogramming towards Pluripotency by Virus-Free Overexpression of Defined Factors

doi: 10.1371/journal.pone.0054754

Figure Lengend Snippet: Balb/C mice HTV injected with 0.9% saline alone, pCX-OKS-2A with (OSKM) and without (OKS) pCX-cMyc in 0.9% saline, or pCAG-GFP in 0.9% saline, at the indicated doses. On day 4, RT-qPCR analysis of hepatocyte extracts was performed. ( a ) Expression levels of the injected reprogramming transcription factors and endogenous pluripotency genes were determined for plasmid dose-escalation (total plasmid dose 50, 75 and 100 µg/animal). All gene expression levels were normalized to the HTV-injected saline group (*p<0.05 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison); ( b ) Expression levels of the injected reprogramming transcription factors and endogenous pluripotency genes with and without inclusion of cMyc . All gene expression levels were normalized to HTV-injected saline group (**p<0.01 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and OKS injected groups, obtained by the analysis of variance and Tukey's pairwise comparison).

Article Snippet: Reprogramming plasmids pCX-OKS-2A encoding OCT3/4, KLF4, SOX2 ; pCX-cMyc encoding CMYC and pCAG-GFP encoding eGFP under the control of CAG promoter (as previously described by Okita et al. ) were obtained from Addgene (USA) as bacterial stabs.

Techniques: Injection, Quantitative RT-PCR, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: In Vivo Cell Reprogramming towards Pluripotency by Virus-Free Overexpression of Defined Factors

doi: 10.1371/journal.pone.0054754

Figure Lengend Snippet: Balb/C mice HTV injected with 0.9% saline alone, 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline, or 150 µg of pCAG-GFP in 0.9% saline and at day 4, livers were collected and frozen tissue sections were stained with anti-OCT4, anti-SOX2 or anti-NANOG antibodies to assess immunoreactivity, or BCIP/NBT to determine ALP activity in the tissue (40x). Scale bars represent 100 µm.

Article Snippet: Reprogramming plasmids pCX-OKS-2A encoding OCT3/4, KLF4, SOX2 ; pCX-cMyc encoding CMYC and pCAG-GFP encoding eGFP under the control of CAG promoter (as previously described by Okita et al. ) were obtained from Addgene (USA) as bacterial stabs.

Techniques: Injection, Staining, Activity Assay

Journal: PLoS ONE

Article Title: In Vivo Cell Reprogramming towards Pluripotency by Virus-Free Overexpression of Defined Factors

doi: 10.1371/journal.pone.0054754

Figure Lengend Snippet: TNG-A mice were HTV injected with 0.9% saline alone, 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline and at days 2, 4, RT-qPCR analysis of hepatocytes was performed to determine the relative gene expression of: ( a ) transfected transcription factors (OKSM) and ( b ) endogenous pluripotency markers. All gene expression levels were normalized to HTV-injected saline group (*p<0.05 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison); ( c ) flow cytometry analysis of GFP positive cells in liver extracts; ( d ) liver tissue frozen and sectioned to image GFP-positive cells with fluorescence microscopy at day 4 (10x).

Article Snippet: Reprogramming plasmids pCX-OKS-2A encoding OCT3/4, KLF4, SOX2 ; pCX-cMyc encoding CMYC and pCAG-GFP encoding eGFP under the control of CAG promoter (as previously described by Okita et al. ) were obtained from Addgene (USA) as bacterial stabs.

Techniques: Injection, Quantitative RT-PCR, Expressing, Transfection, Flow Cytometry, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: In Vivo Cell Reprogramming towards Pluripotency by Virus-Free Overexpression of Defined Factors

doi: 10.1371/journal.pone.0054754

Figure Lengend Snippet: Balb/C mice HTV injected with either 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline or 0.9% saline only. On days 2, 4, 8, 12, 50 and 120 livers and sera were isolated. ( a ) H&E staining of liver sections; ( b ) levels of liver enzymes and ( c ) albumin were analyzed; ( d ) liver sections were PAS stained to determine glycogen storage levels. Representative images were captured with light microscopy (10x). Scale bars represent 100 µm.

Article Snippet: Reprogramming plasmids pCX-OKS-2A encoding OCT3/4, KLF4, SOX2 ; pCX-cMyc encoding CMYC and pCAG-GFP encoding eGFP under the control of CAG promoter (as previously described by Okita et al. ) were obtained from Addgene (USA) as bacterial stabs.

Techniques: Injection, Isolation, Staining, Light Microscopy