Representative (n = 3) Immunofluorescence Imaging of EGFP-TD2.2 Delivery Experiments.

<p>Rows A, C &amp; E show phase (left panels), combined fluorescence (middle panels) and merged images (right panels) of mature human astrocytes, mature human oligodendrocytes and human dermal fibroblasts, respectively. Rows B &amp; D show co-cultured of human astrocytes (unlabelled) and mCherry⁺ human oligodendrocytes. Arrows highlight respective cell types transduced EGFP-TD2.2 recombinant protein.</p

Construction and Optimization of TD domain Purification, and Initial <i>in vitro</i> Screening.

<p>(A) Representation of overlapping PCR primers for generation of novel His-tagged PCR products for cloning to protein expression vectors. (B) Western Blot of expressed pET-Transduction domain (TD) recombinant proteins purified under insoluble (denaturing) conditions (top blot) and soluble (bottom blot) conditions. Predicted proteins are indicated to right of each gel. (C) Oligodendrocyte cultures treated with TD2.2 recombinant protein showing TD2.2 localized to apparent cytosolic (white arrows) and nuclear (red arrows) compartments. Scale bars: 50 µM.</p

Kinetics of EGFP-TD2.2 Recombinant Protein Transduction to Mature Human Oligodendrocytes by Sectional Confocal Microscopy.

<p>A single field of view (single mid-sectional depth) of mCherry⁺ human oligodendrocytes, with hourly differential interference contract (DIC) and mCherry/EGFP images taken following application of 1 µM EGFP-TD2.2 recombinant protein. Arrows track transduction of extracellular EGFP-TD2.2 protein into an oligodendrocyte.</p

Karyotype analysis of ES cells and live offspring derived from vitrified oocytes.

<p>A) Karyotype analysis performed on ViO-ES9 cells revealed a number of abnormalities. The male cell line has a chromosome count of 46. B) Karyotype analysis performed on the mice that were generated from vitrified oocytes revealed normal karyotypes, a representative karyotype for one of the male mice shows a normal 40 XY chromosome count with no abnormalities.</p

Histology and Hematoxylin and Eosin staining of teratoma tissue from ViO-ES9 cells showing differentiation into tissues indicative of the three germ layers (A) including secretory epithelium (i), articular cartilage (ii) and keratinized epithelium (iii).

<p>Immunoflorescent analysis of differentiation into all three germ layers (B): AFP (i); GATA-4 (ii); and NESTIN (iii). Secondary antibodies were labelled with Alexa Fluro® 488 (green) except for GATA-4 which was labelled with Alexa Fluro® 594 (red). Nuclei are stained with DAPI (blue). RT-PCR for Flk-1, VE-Cadherin, PECAM, Vimentin and Nestin (C).</p

Survival, two-cell and blastocyst rates for vitrified mouse oocytes following IVF.

*<p>Fisher's test significantly lower than other treatments (P&lt;0.05);</p>**<p>Fisher's test significantly higher than other treatments within the same column (P&lt;0.001). IVF Data collected from 3 replicates.</p

Survival, vitrified two-cell embryo and live offspring rates from either fresh or vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05).</p

Following in vitro fertilization and culture, both fresh (A1) and vitrified oocytes in 0.1 M trehalose (B1) were capable of developing to the two-cell (A2; B2), four-cell (A3; B3) and blastocyst (A4; B4) stages, respectively.

<p>Furthermore, blastocysts were able to hatch (A5; B5) without chemical or mechanical assistance and some were used to derive embryonic stem cells.</p

Survival, and live offspring rates for vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05). Oocyte and live offspring data collected from 5 and 2 replicates respectively.</p