pmig-oct4 plasmid # 17225  (Addgene inc)

Journal: The Journal of Cell Biology

Article Title: Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency

doi: 10.1083/jcb.201103071

Figure Lengend Snippet: Nucleostemin is essential for maintaining the self-renewal of ESCs. (A) Depletion of NS in ESCs. For A–K, the E14 line of mouse ESCs was transfected with vectors expressing the puromycin resistance gene and either an shRNA targeting Ns or scrambled shRNA. Puromycin selection was initiated 24 h after transfection and continued for 3 d before cells were harvested for subsequent analysis. Untransfected cells were eliminated by 2 d of drug selection. Left, QPCR analysis of Ns mRNA level. Right, Western analysis of NS protein with β-actin as a loading control. The RNA data in A and F were normalized to the expression of Gapdh and are represented as mean ± SEM with n = 3. The data are displayed relative to results with scrambled shRNA transfected controls. The positions of molecular mass standards (in kilodaltons) are shown in A and G. (B) Representative images showing differentiation of ESCs upon NS KD. 4 d after the indicated transfections, NS KD cells became flattened and lost the stereotypical colony morphology and AP staining. Under the same culture conditions, a normal undifferentiated phenotype with distinct colonies strongly expressing AP was maintained in controls with scrambled shRNA. (C) Quantification of undifferentiated, partially differentiated, and fully differentiated ESC colonies 5 d after the indicated transfections. The data in the left panel represent the results of two independent experiments with error bars indicating standard deviations. Approximately 100 colonies were counted for each transfection. The right panel illustrates representative morphology of the three types of colonies used to score the extent of differentiation, as described in Materials and methods. The KD of NS by shRNA-2 was less efficient than that by shRNA-1 (see Fig. S2, C and D for more information). (D) Failure of NS KD cells to form embryoid bodies. 3 d after transfection with shRNAs, ESCs were placed in hanging drops and examined 24–72 h later. The percentage values represent the fraction of hanging drops that formed EBs or remained dispersed after 24 h of culture. (E) Categories of genes whose expression changed in response to KD of NS by Ns shRNA-1 in ESCs. Data were obtained from whole-genome expression analysis of four biological replicates of NS KD cells and scrambled shRNA transfected controls. See Materials and methods and for details. (F) QPCR analysis of RNAs specific for endoderm ( Gata4 and Gata6 ), mesoderm ( Nkx2.5 ), ectoderm ( Fgf5 ), TE ( Cdx2 ), and ESCs ( Zfp42 , Tcl1, Eras , Dppa5 , Sox2 , Utf1 , and Zfp296 ) in NS KD cells and controls. Analysis by QPCR was performed 4 d after the indicated transfections. (G) Western analysis of ESC markers OCT4 and NANOG 4 d after NS KD in ESCs, with β-actin as a loading control. (H) Cell cycle profiles of asynchronously growing ESCs 4 d after transfection with shRNAs. Cells were pulsed with BrdU for 20 min, then fixed and stained with anti-BrdU antibodies and 7-AAD followed by FACS analysis. The percentages of cells in the various phases of the cell cycle are shown. (I) Cumulative BrdU labeling curves. 4 d after the indicated transfections, cells were fed with fresh media containing BrdU every 2 h, and the BrdU labeling index was determined by FACS at the indicated time points. The time necessary to reach the maximum labeling index corresponds to the total cell cycle length minus the length of the S phase (T G2+M+G1 ; ) and was extrapolated by linear regression analysis. One of three independent experiments with comparable results is shown. (J) Cell cycle reentry analysis. Cells with the indicated transfections were synchronized at the G2/M transition at 4 d after transfection with shRNAs, then released to enter S phase, as described in Materials and methods. At subsequent time points, cells were pulsed with BrdU for 20 min and subjected to FACS analysis to measure entry into S phase. Analyses were performed at the time of release from the cell cycle block and 3 h and 9 h afterward. Frequencies indicate percentages of BrdU-positive cells at 9 h after release. (K) DNA replication and apoptosis. 4 d after transfection of ESCs with shRNAs, cells were incubated with BrdU for 22 h, then fixed and stained with anti-BrdU antibodies and Annexin V for subsequent FACS analysis. In total, 93.1% (86.2% + 6.9%) of control transfectants and 88.7% (72.3% + 16.4%) of NS KD cells were positive for BrdU incorporation. Cells that have cycled through the S phase and have become apoptotic during the BrdU labeling period are positive for both BrdU incorporation and Annexin V (6.9% of controls vs. 16.4% of NS KD cells). Bars, 50 µm.

Article Snippet: The human cDNAs for OCT4 , SOX2 , and KLF4 in the pMig vectors were obtained from Addgene.

Techniques: Transfection, Expressing, shRNA, Selection, Western Blot, Control, Staining, Labeling, Blocking Assay, Incubation, BrdU Incorporation Assay

Journal: The Journal of Cell Biology

Article Title: Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency

doi: 10.1083/jcb.201103071

Figure Lengend Snippet: Induction of pluripotent stem cells from mouse mammary epithelial cells by a combination of OCT4 , SOX2 , and NS . (A) Morphology of primary MECs, primary OSN iPS colony 4 wk after transduction, and established OSN iPS clones, the last displaying both characteristic ESC colony morphology and strong staining for AP activity. (B) Expression of pluripotency markers (OCT4 and SSEA1) in OSN iPS cells, as assessed by immunofluorescence microscopy. Nuclei were counterstained with DAPI. The antibodies used in B and C could recognize both human and mouse homologues, but the data likely represent only endogenous mouse proteins due to silencing of the retroviral vectors. (C) Western analysis of NS and OCT4 proteins in OSN iPS cells, OSMK iPS cells, normal ESCs (E14TG2a and D3), and the MECs used to generate the iPS cells. β-Actin was used as a loading control. The positions of molecular mass standards (in kilodaltons) are shown. (D) Detection of transgenes in iPS cells. Genomic DNA extracted from OSN iPS cells, OSMK iPS cells, and MECs used to generate the iPS cells was analyzed by PCR with a forward primer specific for viral vector sequences and a reverse primer for cDNA sequences of human transgenes. Il-2 was used as an internal control. The sizes of the PCR products in D and I are indicated in base pairs. (E) Silencing of transduced genes in iPS cell lines. Gene expression was assessed by QPCR, using primers specific for the transduced human genes. Analyses were performed on uninfected MECs, MECs 48 h after infection with the retroviral vectors, two lines of ESCs (E14TG2a and D3), and two OSN iPS cell lines. The data were normalized to the expression of Gapdh and represent the average of triplicate QPCR analyses. The data are displayed relative to results with newly infected MECs. For E, F, and J, one of three independent experiments with comparable results is shown. (F) Reactivation of endogenous mouse Oct4 , Sox2 , and Nanog in OSN iPS cells. Shown is QPCR analysis with primers detecting transcripts from the respective endogenous mouse loci as opposed to the transduced human cDNAs in OSN iPS cells, two lines of ESCs (E14Tg2a and D3), and the MECs used to generate the iPS cells. Results were normalized to the expression of Gapdh and were the average of triplicate QPCR analyses. The data are shown relative to results with E14Tg2a. (G) Hematoxylin and eosin staining of teratomas generated from OSN iPS cells. Shown is a teratoma containing endoderm (gut-like epithelium), mesoderm (cartilage), and ectoderm (neural tissue). (H) Adult chimeric mouse derived from OSN iPS cells by injection into B6XB6D2 F1 (black) 8-cell-stage embryos and transplantation into pseudopregnant mice. The agouti coat color (arrow) originated from OSN iPS cells derived from129S4 mice. A normal C57BL/6 mouse is shown on the left. (I) Tissue distribution of OSN iPS cells in chimeras. Genomic DNA isolated from indicated organs from an adult chimera derived from OSN iPS cells was analyzed by PCR for the presence of transduced genes as in D. (J) QPCR analysis of endogenous pluripotency markers in early reprogramming cells 12 d after the indicated transductions. The data were normalized to the expression of Gapdh and represent the average of triplicate QPCR analyses. For Sox2 and Nanog , the data are displayed relative to results with MECs transduced with empty vector. For Oct4 , the data are displayed relative to results with MECs transduced with OSK. Bars, 100 µm.

Article Snippet: The human cDNAs for OCT4 , SOX2 , and KLF4 in the pMig vectors were obtained from Addgene.

Techniques: Transduction, Clone Assay, Staining, Activity Assay, Expressing, Immunofluorescence, Microscopy, Retroviral, Western Blot, Control, Plasmid Preparation, Gene Expression, Infection, Generated, Derivative Assay, Injection, Transplantation Assay, Isolation

Journal: The Journal of Cell Biology

Article Title: Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency

doi: 10.1083/jcb.201103071

Figure Lengend Snippet: Induction of pluripotent stem cells from mouse mammary epithelial cells by a combination of OCT4 , SOX2 , and NS . (A) Morphology of primary MECs, primary OSN iPS colony 4 wk after transduction, and established OSN iPS clones, the last displaying both characteristic ESC colony morphology and strong staining for AP activity. (B) Expression of pluripotency markers (OCT4 and SSEA1) in OSN iPS cells, as assessed by immunofluorescence microscopy. Nuclei were counterstained with DAPI. The antibodies used in B and C could recognize both human and mouse homologues, but the data likely represent only endogenous mouse proteins due to silencing of the retroviral vectors. (C) Western analysis of NS and OCT4 proteins in OSN iPS cells, OSMK iPS cells, normal ESCs (E14TG2a and D3), and the MECs used to generate the iPS cells. β-Actin was used as a loading control. The positions of molecular mass standards (in kilodaltons) are shown. (D) Detection of transgenes in iPS cells. Genomic DNA extracted from OSN iPS cells, OSMK iPS cells, and MECs used to generate the iPS cells was analyzed by PCR with a forward primer specific for viral vector sequences and a reverse primer for cDNA sequences of human transgenes. Il-2 was used as an internal control. The sizes of the PCR products in D and I are indicated in base pairs. (E) Silencing of transduced genes in iPS cell lines. Gene expression was assessed by QPCR, using primers specific for the transduced human genes. Analyses were performed on uninfected MECs, MECs 48 h after infection with the retroviral vectors, two lines of ESCs (E14TG2a and D3), and two OSN iPS cell lines. The data were normalized to the expression of Gapdh and represent the average of triplicate QPCR analyses. The data are displayed relative to results with newly infected MECs. For E, F, and J, one of three independent experiments with comparable results is shown. (F) Reactivation of endogenous mouse Oct4 , Sox2 , and Nanog in OSN iPS cells. Shown is QPCR analysis with primers detecting transcripts from the respective endogenous mouse loci as opposed to the transduced human cDNAs in OSN iPS cells, two lines of ESCs (E14Tg2a and D3), and the MECs used to generate the iPS cells. Results were normalized to the expression of Gapdh and were the average of triplicate QPCR analyses. The data are shown relative to results with E14Tg2a. (G) Hematoxylin and eosin staining of teratomas generated from OSN iPS cells. Shown is a teratoma containing endoderm (gut-like epithelium), mesoderm (cartilage), and ectoderm (neural tissue). (H) Adult chimeric mouse derived from OSN iPS cells by injection into B6XB6D2 F1 (black) 8-cell-stage embryos and transplantation into pseudopregnant mice. The agouti coat color (arrow) originated from OSN iPS cells derived from129S4 mice. A normal C57BL/6 mouse is shown on the left. (I) Tissue distribution of OSN iPS cells in chimeras. Genomic DNA isolated from indicated organs from an adult chimera derived from OSN iPS cells was analyzed by PCR for the presence of transduced genes as in D. (J) QPCR analysis of endogenous pluripotency markers in early reprogramming cells 12 d after the indicated transductions. The data were normalized to the expression of Gapdh and represent the average of triplicate QPCR analyses. For Sox2 and Nanog , the data are displayed relative to results with MECs transduced with empty vector. For Oct4 , the data are displayed relative to results with MECs transduced with OSK. Bars, 100 µm.

Article Snippet: The human cDNAs for OCT4 , SOX2 , and KLF4 in the pMig vectors were obtained from Addgene.

Techniques: Transduction, Clone Assay, Staining, Activity Assay, Expressing, Immunofluorescence, Microscopy, Western Blot, Plasmid Preparation, Infection, Generated, Derivative Assay, Injection, Transplantation Assay, Isolation