Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.

During mouse eye development, all retinal cell types are generated from the population of retina-committed progenitors originating from the neuroepithelium of the optic vesicle. Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited. Here we report generation of the mRx-Cre BAC transgenic mouse line in which the expression of Cre recombinase is controlled by regulatory sequences of the mouse Rx gene, one of the earliest determinants of retinal development. When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup. Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development. Moreover, since eye organogenesis is dependent on the inductive signals between the optic vesicle and head surface ectoderm, the inductive ability of the optic vesicle can be analyzed using mRx-Cre transgenic mice

Solid-phase extraction based on hydrophilic interaction liquid chromatography with acetone as eluent for eliminating matrix effects in the analysis of biological fluids by LC-MS

Analysis of drugs and metabolites in biological matrices such as blood or plasma by LC-MS is routinely challenged by the presence of large quantities of competing molecules for ionization in soft ionization sources, such as proteins and phospholipids. While the former can easily be removed by protein precipitation, pre-analytical extraction of the latter is necessary because they show very high retention in reversed-phase LC resulting in long analysis times or in ion suppression effects when not eluted before the next runs. A novel HILIC-based SPE approach, making use of silica cartridges and of acetone as organic solvent, is introduced as a potent alternative to current commercial methods for phospholipid removal. The methodology was developed and tested for a broad polarity range of pharmaceutical solutes (log P from 0 to 6.6) and broad applicability can therefore be envisaged

HOSPITALIZATION BURDEN IN CHILDREN WITH CHRONIC KIDNEY DISEASE (CKD): FINDINGS FROM THE 4C STUDY

WOS: 000408418900455

CORRELATION OF ACOUSTIC RADIATION FORCE IMPULSE (ARFI) ELASTOGRAPHY WITH HISTOLOGICAL FINDINGS IN PEDIATRIC RENAL TRANSPLANT PATIENTS

WOS: 000408418900055

Schematic representation of <i>Cre</i> recombinase-expressing transgenic mouse lines used in this study.

<p>(A) The <i>Rx-Cre</i>; with a 4-kb DNA fragment upstream of the medaka <i>Rx3</i> gene driving <i>Cre</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063029#pone.0063029-Swindell1" target="_blank">[12]</a>. (B) To generate <i>MB31-Cre</i>, the 4-kb DNA fragment upstream of the medaka <i>Rx3</i> gene was linked to the coding region of <i>Cre</i> recombinase. The <i>IRES</i> sequence was used to connect <i>Cre</i> expression with EGFP to monitor the transgene expression. (C) To generate <i>mRx-Cre BAC</i>, a BAC containing 200 kb covering the <i>Rx</i> locus was modified by BAC recombineering. The <i>Cre</i> coding region (Cre-pA) was inserted into the <i>Rx</i> translational initiation start site (ATG). The exons are indicated with black boxes.</p

<i>In vivo</i> activity of <i>Rx-Cre</i>, <i>MB31-Cre</i> and <i>mRx-Cre</i> transgene products assessed using the <i>ROSA26R</i> line.

<p>Whole-mounts (A–F) or coronal sections (A’–F’) were stained with X-gal at indicated stages to show the Cre activity in the eye primordium (white arrowheads). Surface ectoderm and developing lens are indicated with dashed lines. SE – surface ectoderm; OV – optic vesicle; RPE – retinal pigmented epithelium; RE – retina; vf – ventral forebrain.</p

Activity of <i>Rx-Cre</i>, <i>MB31-Cre</i> and <i>mRx-Cre</i> in the eye primordium analyzed using the <i>ROSA26R-EYFP</i> reporter line.

<p>(A–C) Whole-mounts showing <i>EYFP</i> expression (green) in the overall embryo at E10.5. (A’–C’) Coronal sections through the eye region co-stained with DAPI (blue) showing Cre activity in the retina, retinal pigmented epithelium and invaginating lens pit (dashed line) at E10.5.</p

Abnormal Lens Morphogenesis and Ectopic Lens Formation in the Absence of β-Cateinin Function

β-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of β-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate β-catenin in developing lens and retina. Inactivation of β-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that β-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of β-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of β-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, β-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate

The <i>mRx-Cre</i> activity in the eye, forebrain and hypothalamus analyzed using the <i>ROSA26R</i> reporter line.

<p>Whole-mounts or sections were stained with X-gal at indicated stages to show the <i>mRx-Cre</i>-mediated Cre activity. (A) The X-gal⁺ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.</p