<i>CE4-lacZ</i> displays expression in a subset of <i>Dlk1</i>-expressing tissues.

<p>(A) Diagram of <i>lacZ</i> expression constructs used to produce <i>CE4-lacZ</i> and <i>CE8-lacZ</i> transgenic embryos. The arrow represents the direction of transcription, and CE stands for conserved element 4 or 8. (B–D) Ventral, dorsal and lateral views, respectively, of a representative whole mount transgenic embryo at e13.5. (E) Schematic representation of sagittal section planes used in these images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Kaufman1" target="_blank">[64]</a>. (F–M) Sagittal sections of embryos under low magnification (F, I, L) and high magnification (G, H, J, K, M). All sections are oriented with anterior on top and dorsal to the left. Expression was seen in (F–H) intercostal muscle, body wall muscle, and ribs; (I, J) dorsal root ganglia; (I, K) intrinsic tongue muscle; (L, M) thymus. The embryo shown in (F) displays <i>lacZ</i> expression in the pituitary gland and trigeminal ganglion, both sites of endogenous <i>Dlk1</i> expression, but this pattern was not seen in other embryos carrying <i>CE4-lacZ</i>. Scale bars represent 100 µm.</p

<i>Dlk1</i> is expressed but not imprinted from the 127H5 transgene.

<p>(A) Schematic of the <i>Dlk1</i>-<i>Meg3</i> BAC clones used to generate transgenic mice; the <i>28G5</i> transgene was described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Yevtodiyenko2" target="_blank">[46]</a>. The 127H5 BAC was linearized at a unique <i>Cla</i>I site. (B, C) Representative Northern blots for <i>Dlk1</i> mRNA in midgestation wild type (WT) and heterozygous transgenic (Tg) embryo (B) and placenta (C), after paternal (<i>127H5^Pat</i>) and maternal (<i>127H5^Mat</i>) inheritance. The mouse <i>β-actin</i> gene was used as a loading control. (D) Quantitative Northern data for blots shown in B & C; expression is normalized to <i>β-actin</i>. Gray bars represent wild type samples and black bars represent <i>127H5</i> transgenic samples upon paternal (<i>127H5^Pat</i>) or maternal (<i>127H5^Mat</i>) transmission in crosses to Cg12. (E) Direct sequencing assay for <i>Dlk1</i> imprinting in wild type (WT) and heterozygous transgenic (Tg) F₁ embryos after paternal (<i>127H5^Pat</i>) and maternal (<i>127H5^Mat</i>) inheritance. D indicates wild type animals carrying only the <i>M. domesticus</i> allele, while D×C or C×D indicates offspring of crosses to the Cg12 line carrying a <i>M. castaneus</i> allele, with the female genotype listed first. (F) Quantitative Northern blot analysis for <i>Dlk1</i> mRNA in 3–4 week old <i>127H5</i> tissues. Expression is normalized to <i>β-actin</i>, and each bar represents 8–10 animals. Gray bars represent wild type samples and the black bars represent <i>127H5</i> transgenic samples upon paternal (Pat) or maternal (Mat) transmission. In all figures asterisks indicate p≤0.05.</p

Embryonic expression of <i>CE4-lacZ</i>, <i>CE8-lacZ</i> and <i>Dlk1.</i>

<p>+ indicates expression; – indicates no expression.</p

Sequence conservation in the <i>Dlk1</i> upstream region.

<p>The sequence upstream of <i>Dlk1</i> was analyzed for regions of conservation across multiple species. The conserved elements chosen for further analysis are numbered CE1 to CE9. The region being displayed corresponds to the July 2007 mouse genome assembly; assembly dates for other species are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#s2" target="_blank">Methods</a> (adapted from the UCSC Genome Browser).</p

<i>CE8-lacZ</i> displays expression in a subset of <i>Dlk1</i>-expressing tissues

<p>. (A–C) Ventral, dorsal and lateral views, respectively, of a representative whole mount transgenic embryo at e13.5. (D) Schematic representation of sagittal section planes used in these images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Kaufman1" target="_blank">[64]</a>. (E–Q) Sagittal sections of embryos under low magnification (E, G, J, L, N, P) and high magnification (F, H, I, K, M, O, Q). All sections are oriented with anterior on top and dorsal to the left. Expression was seen in (E, F) skeletal muscle of the limb, (G, H) intervertebral cartilage; (G, I–K) ribs and costosternal junctions; (L, M) chromaffin cells of the adrenal gland; (N, O) dorsal root ganglia; (P, Q) spinal cord. Scale bars represent 100 µm.</p

Transcriptional activation by conserved elements is cell line-dependent.

<p>(A) Luciferase expression plasmids used to assay enhancer function in cell culture. “<i>Dlk1</i> Promoter” signifies the <i>Dlk1</i> basal promoter and CE signifies the individual conserved elements tested. Plasmid pGL3-B contains the promoterless luciferase gene, pGL3-<i>Dlk1</i>P contains the endogenous <i>Dlk1</i> promoter upstream of luciferase and pGL3-C contains the SV40 promoter/enhancer upstream of luciferase. (B) Enhancer activity observed in the NIH-3T3, C2C12, SVR and Y-1 cell lines. Expression is normalized to pGL3-<i>Dlk1</i>P for each cell type; the results are presented as mean ± SEM. P-values relative to pGL3-<i>Dlk1</i>P expression are indicated by *, p≤0.05; #, p≤0.01 (n = 3) (Student’s t-test).</p