PCR primers.

<p>PCR primers.</p

Fluorescent immunohistochemistry of articular cartilage.

<p>Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA<i>later</i>/EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.</p

Quantitative PCR.

<p>Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA<i>later</i>/EDTA at pH 9.2 or RNA<i>later</i>/EDTA at pH5.2 was analyzed for <i>Col2a1</i> (A) or <i>Rpl10</i> (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.</p

<i>In situ</i> hybridization for <i>Prg4</i> and <i>Col2a1</i>.

<p>Cryosections from tibia decalcified with EDTA (A,B) or RNA<i>later</i>/EDTA at pH5.2 (C–F) were hybridized with DIG-labeled <i>Prg4</i> antisense (A,C) or sense (B,D) RNA probes, and against ³⁵S-labeled <i>Col2a1</i> antisense (E) or sense (F) RNA probes for <i>Col2a1</i>. Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).</p

Analysis of RNA integrity.

<p>Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA<i>later</i>/EDTA at pH 9.2 (C, D) or RNA<i>later</i>/EDTA at pH5.2 (E, F).</p

<i>Sox3</i>-26ala cells cause pituitary defects indistinguishable from <i>Sox3</i>-null cells.

<p>WT, <i>Sox3</i>+/− or <i>Sox3</i>-26ala<->WT chimeras were cut sagittally at 11.5 dpc (A) or 13.5 dpc (B) and immunostained for SOX3 and NEO expression. Percentage chimerism for each embryo in (A) and (B) was determined by qPCR as outlined in the methods. ISH for <i>Neo</i> on adjacent sections at 11.5 dpc confirmed the identification of mutant cells within the ventral diencephalon (A). Examples at 11.5 dpc show the infundibulum (I) appears unaffected in a 5% chimera, shallow in a 20% chimera and absent in a 75% chimera that also displayed a Rathke's Pouch (*) that had failed to detach from the oral ectoderm. At 13.5 dpc, heterozygous and high percentage (65%) chimeric embryos displayed a distorted infundibulum (I) with a lobular edge (arrow heads) and a branched Rathke's Pouch (*). Low percentage chimeras (20%) look similar to WT (0%). C) Phase micrographs of 13.5 dpc coronal sections through the developing pituitary show a broadening at the base of the third ventricle in chimeras (arrows). Chimerism for embryos shown in (C) was determined based on immunoreactivity for NEO in adjacent sections (data not shown).</p

Generation of <i>Sox3</i>-26ala ES cells.

<p>Scale representation of the <i>Sox3</i> locus, targeting vector and recombinant alleles (A). Probing of <i>BglII</i> digested DNA from ES cell clones with the 5′ probe yielded an 8.8 kb fragment from the WT locus and a 5.9 kb fragment when the <i>Neo</i> cassette was recombined into the <i>Sox3</i> locus (<i>Sox3</i>-26ala or <i>Neo</i>). B) Representative Southern blot of 3 clones including a targeted clone (<i>Sox3</i>-26ala-3) is shown. C) PCR using primers spanning the alanine expansion (red arrows in A) was used to distinguish whether targeted clones carried the expansion and gave a 219 bp product instead of 186 bp as seen in WT.</p

SOX3-26ala from mouse and human retains transactivation activity.

<p>A) COS-7 cells were transfected with pcDNA3.1 expression vector containing either mouse <i>Sox3</i>, human <i>SOX3</i>, mouse <i>Sox3</i>-26ala, human <i>SOX3</i>-26ala or an empty vector control. Values represent mean normalised luciferase values plus standard deviation of four independent experiments measured 48 hours after transfection. Student's T-tests (two tailed, unequal variance) of SOX3-26ala from human or mouse compared to empty vector control show a statistically significant increase in luciferase activity. B) Nuclear protein lysates prepared from duplicate plates 48 hours after transfection show that less SOX3 is detected in the nucleus of cells expressing both mouse and human SOX3-26ala. pcDNA3.1-EGFP transfected cells were used as a control and prepared for both nuclear protein and whole cell extracts (WCE). Blotting for Histone H3, indicates equal loading and blotting for α-Tubulin shows an absence of cytoplasmic contamination in nuclear preparations. Transfection efficiency was determined by co-transfecting EGFP and counting positive cells prior to harvesting and found to be equal for all plasmids.</p

Failure of <i>Sox3</i>-26ala targeted ES clones to transmit through the germline.

<p>Failure of <i>Sox3</i>-26ala targeted ES clones to transmit through the germline.</p

Residual nuclear SOX3-26ala protein rescues a gastrulation defect of <i>Sox3</i>-null <-> WT chimeric embryos.

<p><i>Sox3</i>-26ala <-> WT chimeras are normal at 7.5 dpc (gastrulation) unlike <i>Sox3</i>-null <-> WT chimeras. A total of 15 <i>Sox3</i>-flox<-> WT ES chimeras, 31 <i>Sox3</i>-null<-> WT chimeras and 21 <i>Sox3</i>-26ala<-> WT chimeras were blind scored by two independent operators as morphologically normal or abnormal. The average score for each embryo was used to plot the percentage of abnormal embryos for each condition and chi squared analysis was performed with <i>Sox3</i>-flox<-> WT embryos used to set expected outcomes. Significantly more <i>Sox3</i>-null<-> WT chimeras were abnormal (p = 0.0001) while <i>Sox3</i>-26ala<-> WT chimeras did not deviate from expected (p = 0.95). An example of normal morphology is shown for <i>Sox3-</i>flox<-> WT and <i>Sox3</i>-26ala<-> WT chimeras and an abnormal <i>Sox3</i>-null<-> WT chimera is also shown that exhibits distortion of the ectodermal layer and apparent expansion of cells at the primitive streak and the adjacent extra-embryonic region.</p