Apparatus for embryo manipulation, electroporation and image capture.

<p>(A) A stereoscopic microscope for embryo manipulation. (B) Chamber-type electrodes with 1-mm gap used in this experiment. (C) Electro Cell Manipulator for generating electric pulses. (D) Nikon DS-Ri1 digital camera for image capture.</p

Effects of <i>Oct4</i>-targeting morpholinos on early development.

<p>(A) Blastocyst development following electroporation of zygotes with customized <i>Oct4</i> morpholinos at different concentrations and cultured for 3.5 d. (B) Blastocyst development following electroporation of zygotes with different morpholinos with a concentration of 0.4 mM. (C) Morphological appearance of electroporated zygotes obtained from the control and <i>Oct4</i>-MOs groups after being cultured for 3.5 d. The original magnification was ×100. (D) Nuclear Oct4 expression is absent in <i>Oct4</i>-MOs electroporated embryos but present in embryos obtained from the control groups. Each sample was counterstained with Hoechst 33342 to visualize DNA (blue). The original magnification was ×200.</p

Electroporation of <i>Oct4</i>-specific shRNA expression vectors.

<p>(A) Blastocyst development following electroporation of zygotes with custom <i>Oct4</i>-specific shRNA expression vectors at different concentrations and cultured for 3.5 d. (B) Blastocyst development following electroporation of zygotes with different shRNA expression vectors with a concentration of 100 µg/ml. (C) Morphological appearance of electroporated zygotes obtained from the control and <i>Oct4</i>-shRNA groups after being cultured for 3.5 d. Morphology (top) and fluorescence (bottom). The original magnification was ×100.</p

Plasmid DNA and morpholinos were efficiently introduced into mouse preimplantation embryos by electroporation.

<p>1-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos and morulas were electroporated with pIRES2-AcGFP1-Nuc (A) or the lissamine conjugated morpholinos (B) and cultured for 24 h. pIRES2-AcGFP1-Nuc and morpholinos introduce, revealed by green fluorescent protein and lissamine fluorescence, respectively, is efficient. 4-cell embryos and 8-cell embryos were all developed from the electroporated 2-cell embryos. Original magnification was ×200.</p

Electroporation conditions and efficiencies for DNA and morpholinos.

a<p>The electroporation parameters contain voltage, pulse duration, number of pulses and repeats. Three replicate experiments were performed per embryonic stage.</p

Functional activity of wildtype and mutant Ipr1 in regulation assays.

<p>(A) A representative blot from a western blot analysis demonstrating the expression of the different mutant proteins. (B) Results from an assay measuring the gene repression induced by Ipr1. The expressions of Il10, Pmp2, and Ccl2 were determined by qPCR after the transient transfection of RAW264.7 cells with wildtype Ipr1 or various Ipr1 mutants. (C) Results from an assay measuring the gene activation induced by Ipr1. The expressions of Il6, Ccnd2, and Pdcd2 were determined by qPCR after the transient transfection of RAW264.7 cells with wildtype Ipr1 or various Ipr1 mutants. (D) RAW 264.7 cells were transfected with empty vector as control, wildtype Ipr1, or Ipr1-R424A-K429A. After 12 h, each group of transfected cells was incubated in the absence or presence of H37Ra. Apoptotic cells were evaluated by Annexin-V staining followed by flow cytometric analysis. The apoptotic cell rate, which is presented in the right panel, was quantified by the following algorithm: percentage of Annexin-V+ and PI− cells in the presence of H37Ra minus the percentage of Annexin-V+ and PI− cells in the absence of H37Ra. Data represent the mean ± SD of three independent experiments. Two asterisks, p < 0.01.</p

Functional validation of the Ipr1 cNLSs.

<p>(A) Schematic representation of Ipr1 protein. The positions of two cNLSs (black rectangles) identified by web-based program analyses are indicated, and the sequence of each cNLS is presented below. (B) Schematic representation of the EGFP-GST construct used in the nuclear import assessment. Each Ipr1 cNLS sequence was cloned into downstream of the EGFP-GST. The fluorescence images show representative samples of NIH3T3 cells transfected with each of the expression plasmids for 24 h. The cell nuclei were counterstained with DAPI. (C) Schematic representation of Ipr1 mutants bearing deletions of cNLS1, cNLS2, or both. The fluorescence images show representative results from the nucleocytoplasmic localization of these Ipr1 mutants in NIH3T3 cells. The cell nuclei were counterstained with DAPI.</p

Structure and sequence analysis of Ipr1 protein.

<p>(A) Domain structure of Ipr1, Sp100 and Sp140 proteins. Ipr1 protein consists of a conserved Sp100-like domain and a SAND domain containing a DNA-binding motif. (B) Comparison of the amino acid sequences of Sp100-like and SAND domains among Ipr1, Sp100, and Sp140. Identical amino acids are indicated by an asterisk, and strongly or weakly similar amino acids are indicated by a colon or a period. Some of the conserved amino acid residues are marked by shaded boxes. The percentage of identical residues and similarity between domains of Ipr1, Sp100 and Sp140 is calculated in the followed tables.</p

Assessment of the Sp100-like and SAND domain necessities for the dimerization and ND-targeting of Ipr1.

<p>(A) Lysates of 293FT cells expressing p3×FLAG-Ipr1 with HA-Ipr1, HA-Ipr1-ΔSp100, or HA-Ipr1-ΔSAND were immunoprecipitated (IP) with anti-FLAG antibodyand detected by western blot (WB) with anti-HA antibody. Input represents 10% of the starting material. (B) 293FT cells were transfected with p3×FLAG-Ipr1. After 24 h, the cells were collected and the resulting cell extracts were subjected to chemical cross-linking by using DSS in DMSO or to DMSO alone as a control. The protein samples were then analyzed by western blot assays using anti-FLAG antibody. (C) RAW264.7 cells were transfected with EGFP-fused Ipr1-WT (top), Ipr1-ΔSAND (middle), or Ipr1-ΔSp100 (bottom). Immunofluorescence was performed using rat anti-PML antibody (red). Merged images show the co-localization of these proteins in yellow.</p

Schematic representation of the potential mechanism for Ipr1 nuclear import and transcriptional regulation.

<p>(A) A model illustrating the ability of Ipr1 forms homo/hetero dimers and transports into the nuclear by binding with importin protein. (B) The hypothesized regulation mode of Ipr1 on both transcriptional inhibition and activation.</p