Quantitative RT-PCR for Oct4 in whole-culture (*) and micro- (**) XEN-P cell line samples.

<p>Samples (numbers in brackets) were RNA-extracted, RNA preparations were DNAse-treated and quantified in duplicate by real-time RT-PCR using dual-specific (mouse = rat) primers; controls without reverse transcriptase did not yield a product. Data (Means±SEM) were normalized to Hprt mRNA and expressed as fold of the level in ES cells, i.e. ES cell level is set as 1.</p>A<p>, ^B, two groups of microsamples with high and moderate Oct4 mRNA expression, respectively. Two experiments labeled ^C were corrected for feeder cell RNA; the other measurements are slight underestimates.</p

Contributions of rat XEN-P cell lines to postimplantation embryos.

<p>Representative fluorescence (A–C) and bright field (A'–C') photographs demonstrating in vivo contributions of microinjected rat cells to (A, A') parietal yolk sac of an 11.5 dpc rat conceptus (inset showing magnification); (B, B') visceral endoderm of an 8.5 dpc rat conceptus; (C, C') visceral endoderm (arrowheads; one patch magnified in inset) of an ∼7 dpc mouse conceptus. Pregnancy timing is distorted by the embryo manipulations and therefore only approximate.</p

List of primers used in this study.

<p>List of primers used in this study.</p

Lineage marker analysis during the evolution of colonies growing after low-density plating of a rat XEN-P cell line (RX1).

<p>(A) Double staining for Oct4 (green) and Gata6 (red); (B) Double staining for Oct4 (green) and Gata4 (red); (C) Double staining for Oct4 (red) and SSEA1 (green); (D) Staining for SSEA3; (E) Staining for Laminin B; (F) Staining for Collagen 4. BF, bright field. RX1 cells were plated at 25–50 cells/cm², and at different time points, the resulting colonies were stained with the indicated antibodies and counterstained with DAPI. Controls omitting primary antibodies were negative and are not shown. The speed of colony evolution varied somewhat between experiments, resulting in “Young” colonies at days 2–3, “Intermediate” colonies at days 3–5, and “Mature” colonies at days 5–7 (day 0 = day of plating).</p

Properties of rat blastocyst outgrowths.

<p>(A) Phase contrast photographs showing stages of WKY rat blastocyst outgrowths kept on mitomycin-treated primary rat embryo fibroblasts (PREF). The outgrowths were initially smooth and compact (left), but converted to XEN morphology (right) ∼10 days after blastocyst plating if not passaged, or a few days later if mechanically disaggregated into smaller clumps. Regardless of when the conversion occurred, it was fast (<24 hours) and went through a stage of intermediate morphology (middle). (B) Loss and re-expression of Oct4 mRNA in WKY rat blastocyst outgrowths. In these experiments, the outgrowths were not passaged and showed compact, smooth morphology before day 10, but XEN morphology thereafter. At the indicated days, the outgrowths were individually harvested for RT-PCR analysis, using rat-specific primers for Oct4 and hypoxanthine phosphoribosyl transferase (Hprt) cDNAs. The Oct4 and Hprt cDNAs were amplified in the same reaction; none of the primers amplified intronless products from genomic DNA (not shown). No amplification was achieved when using mouse-specific primers (not shown). Day 0 = blastocyst; W, water control. (C) Semi-quantitative assessment of Oct4 mRNA level. Rat blastocysts (E4.5, strain WKY), XEN-P line RX1, primary XEN-like blastocyst outgrowths (strain WKY), rat embryo fibroblast line Li 1 (feeder for RX1), and PREF (feeder for primary rat cells) were analyzed for Oct4 and Hprt mRNAs by subjecting 10-fold serial dilutions of the RT reactions to PCR. (D) LIF effect (1,000 u/ml) on the formation of secondary XEN-like cell colonies from primary rat blastocyst outgrowths (WKY). Primary cells were seeded at ∼100–500 cells/well onto feeder line Li 1. 6 independent experiments. Similar results were obtained with rat strain BDIX.</p

Growth behavior and comparative embryonic lineage marker analysis of rat XEN-P cell lines.

<p>(A) Phase contrast photo showing characteristic morphology of rat XEN-P cell lines growing on rat embryo fibroblast feeder. Colonies obtained by low-density plating typically contained round, refractile cells at their fringes and epithelial cells inside (inset). (B) Representative photos illustrating that LIF (1000 u/ml) increased colony diameter and frequency (crystal violet staining) (line RX1). Similar results were obtained with line RX2 (strain BDIX). (C) RT-PCR analysis showing that rat XEN-P cell lines exhibit a mixed embryonic lineage marker profile. Rat XEN-P cell lines (RX1, RX2, RX5) were compared with mouse XEN cell lines (MX4, MX6), a mouse ES cell line (D3), a trophectoderm-like rat cell line (B10), a rat embryo fibroblast cell line (Li1) used as feeder for the XEN-P cell lines, and primary mouse embryo fibroblasts (MEF) used as feeders for mouse XEN and ES cells. Lines D3 and B10 have been described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone.0007216-Doetschman1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone.0007216-EppleFarmer1" target="_blank">[13]</a>. 2 µg of RNA per sample were reverse-transcribed or not (-RT), followed by PCR using dual-specific (rat = mouse) primers. For Gata6, Foxa2, and Dab2, two dilutions of the RT reaction were subjected to PCR for semi-quantitative comparison. (D) Western blot analysis of XEN-P (RX1), mouse XEN (MX4), and feeder (MEF, Li1) cell lines. 40 µg of cell protein were loaded per lane. (E) Northern blot analysis of XEN-P (RX1), mouse XEN (MX4), mouse ES (D3), and feeder (MEF, Li1) cell lines. 5 µg of total RNA were loaded per lane. (F) Western blot analysis for Oct4 in rat XEN-P (RX1), mouse XEN (MX4), mouse ES (D3), and feeder (MEF, Li1) cell lines, using a monoclonal anti-Oct4 antibody. 50 µg (top) or the indicated amounts (bottom) of cell protein were loaded. RX1 samples from two passages (P39, P40) were analyzed (bottom). Similar results were obtained with a polyclonal antibody (not shown). (G) Transient expression of mouse Oct4 gene-based LacZ reporter gene GOF9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone.0007216-Yeom1" target="_blank">[53]</a> by rat XEN-P and mouse ES but not mouse XEN cell lines. Histochemical stainings of lines D3, MX4, and RX1 (similar results were obtained with line RX2). Non-transfected cells did not show LacZ staining (not shown). When comparing the frequencies of reporter gene expression in mouse ES vs. rat XEN-P cell lines, keep in mind that only a subpopulation in the rat cell lines highly expresses the endogenous <i>Oct4</i> gene (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007216#pone-0007216-g003" target="_blank">Fig. 3</a>).</p

Continued proliferation and preferential accumulation of SSEA1-positive cells in old rat colonies derived from rat XEN-P cells.

<p>Two magnifications of a representative 16-days old colony (line RX1) are shown. Bright field (left), immunofluorescence (middle), and nuclear stain (right). Control omitting primary antibody was negative and is not shown.</p

List of antibodies used in this study.

*<p>SC, Santa Cruz Biotechnology, Inc.; Ab, Abcam plc; JI, Jackson Immunoresearch; DHSB, Developmental Studies Hybridoma Bank, University of Iowa.</p