Characterization of the Frizzled10‐CreER™ transgenic mouse: An inducible Cre line for the study of Cajal‐Retzius cell development

Cajal‐Retzius cells are an enigmatic class of neurons located in the most superficial layer of the cerebral cortex, and they play an important role in cortical development. Although many studies have indicated that CR cells are involved in regulating cell migration and cortical maturation, the function of these cells is still not fully understood. Here we describe an inducible Cre mouse line in which CreER? is driven by the promoter for the Wnt receptor Frizzled10. Consistent with our previous studies on Frizzled10 expression and transgenic mouse lines using the Frizzled10 promoter, we found that in the developing telencephalon, Cre was mainly detected at the cortical hem, the largest source of CR cells. By crossing the Cre line to R26R reporter mice and injecting tamoxifen at different time points, we were able to detect via X‐gal staining CR cells produced from the cortical hem at distinct stages during development. Thus, this transgenic Cre mouse line is a valuable tool for studying the molecular and cellular mechanisms of CR cell development. genesis 47:210–216, 2009. © 2009 Wiley‐Liss, Inc.     

Generation and characterization of PDGFRα‐GFPCreERt2 knock‐In mouse line

Platelet‐derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα‐expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα‐expressing cells in vivo, we generated a knock‐in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand‐binding domain (ER^T2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα‐expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock‐in mouse line generated here could be useful for studying PDGFRα‐expressing cells and their descendants in vivo at various stages of development. genesis 53:329–336, 2015. © 2015 Wiley Periodicals, Inc.     

Mouse lines with photo‐activatable mitochondria to study mitochondrial dynamics

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines withphoto‐activatable mitochondria (PhAM). In the PhAM ^floxed line, a mitochondrially localized version of the photo‐convertible fluorescent protein Dendra2 (mito‐Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP‐flanked termination signal. Expression of Cre in PhAM ^floxed cells results in bright mito‐Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM ^floxed line expresses mito‐Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM ^excised), the expression of mito‐Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo‐conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states. © genesis 1–11, 2012. © 2012 Wiley Periodicals, Inc.     

Sox9‐expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons

Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell‐lineage tracing and gene‐function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9^CreERT2/+;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9‐expressing tissues. To determine the suitability of this mouse line for developmental stage‐specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9‐expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9‐expressing cells. genesis 48:635–644, 2010. © 2010 Wiley‐Liss, Inc.     

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

Centrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue‐specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP‐Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre‐mediated excision, EGFP‐Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real‐time imaging, and showed that EGFP‐Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP‐Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286–296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

Capn8 promoter directs the expression of Cre recombinase in gastric pit cells of transgenic mice

Gastric pit cells are high‐turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8‐Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca²⁺‐regulated cysteine protease calpain‐8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8‐Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4^Co/Co). Multiple‐tissue PCR and LacZ staining demonstrated that Capn8‐Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8‐Cre mouse line described here could be used to dissect gene function in gastric pit cells. genesis 47:674–679, 2009. © 2009 Wiley‐Liss, Inc.     

A Cre‐inducible fluorescent reporter for observing apical membrane dynamics

The ability to image living tissues with fluorescent proteins has revolutionized the fields of cell and developmental biology. Fusions between fluorescent proteins and various polypeptides are allowing scientists to image tissues with sub‐cellular resolution. Here, we describe the generation and activity of a genetically engineered mouse line expressing a fusion between the green fluorescent protein (GFP) and the apically localized protein Crumbs3 (Crb3). This reporter drives Cre‐inducible expression of Crb3–GFP under control of the EF1a regulatory domains. The fusion protein is broadly expressed in embryonic and adult tissues and shows apical restriction in the majority of epithelial cell types. It displays a variably penetrant gain of function activity in the neural tube. However, in several cell types, over‐expression of Crb3 does not appear to have any effect on normal development or maintenance. Detailed analysis of kidneys expressing this reporter indicates normal morphology and function highlighting the utility for live imaging. Thus, the EF1a^Crb3–GFP mouse line will be of broad use for studying membrane and/or tissue dynamics in living tissues. genesis 53:285–293, 2015. © 2015 Wiley Periodicals, Inc.     

Characterization of a novel transgenic mouse line expressing Cre recombinase under the control of the Cdx2 neural specific enhancer

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal‐related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R‐YFP reporter mice have indicated that the Cdx2NSE‐Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double‐transgenic embryos has revealed that this novel Cre‐driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso‐ventral axis (including pre‐migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non‐cranial neural crest derivatives of double‐transgenic embryos. Therefore, we believe that the Cdx2NSE‐Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 51:777–784. © 2013 Wiley Periodicals, Inc.     

Development of a general‐purpose method for cell purification using Cre/loxP‐mediated recombination

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general‐purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock‐in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP‐mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1‐Cre‐transgenic (tg) embryos almost equally as efficiently as from Nr5a1‐hCD271‐tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh‐Cre‐tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. genesis 53:387–393, 2015. © 2015 Wiley Periodicals, Inc.     

A Tlx2‐Cre mouse line uncovers essential roles for hand1 in extraembryonic and lateral mesoderm

Hand1 regulates development of numerous tissues within the embryo, extraembryonic mesoderm, and trophectoderm. Systemic loss of Hand1 results in early embryonic lethality but the cause has remained unknown. To determine if Hand1 expression in extraembryonic mesoderm is essential for embryonic survival, Hand1 was conditionally deleted using the HoxB6‐Cre mouse line that expresses Cre in extraembryonic and lateral mesoderm. Deletion of Hand1 using HoxB6‐Cre resulted in embryonic lethality identical to systemic knockout. To determine if lethality is due to Hand1 function in extraembryonic mesoderm or lateral mesoderm, we generated a Tlx2‐Cre mouse line expressing Cre in lateral mesoderm but not extraembryonic tissues. Deletion of Hand1 using the Tlx2‐Cre line results in embryonic survival with embryos exhibiting herniated gut and thin enteric smooth muscle. Our results show that Hand1 regulates development of lateral mesoderm derivatives and its loss in extraembryonic mesoderm is the primary cause of lethality in Hand1‐null embryos. genesis 48:479–484, 2010. © 2010 Wiley‐Liss, Inc.     

Spatiotemporal regulation of switching front–rear cell polarity

Bacterial cells are spatiotemporally highly organised with proteins localising dynamically to distinct subcellular regions. Motility in the rod-shaped Myxococcus xanthus cells represents an example of signal-induced spatiotemporal regulation of cell polarity. M. xanthus cells move across surfaces with defined front–rear polarity; occasionally, they invert polarity and, in parallel, reverse direction of movement. The polarity module establishes front–rear polarity between reversals and consists of the Ras-like GTPase MglA and its cognate GEF and GAP, that all localise asymmetrically to the cell poles. The Frz chemosensory system constitutes the polarity inversion module and interfaces with the proteins of the polarity module, thereby triggering their polar repositioning. As a result, the polarity proteins, over time, toggle between the cell poles causing cells to oscillate irregularly. Here, we review recent progress in how front–rear polarity is established by the polarity module and inverted by the Frz system and highlight open questions for future studies.     

Labeling of BSA and imaging of mouse T‐lymphocyte as well as mouse spleen tissue by l‐glutathione capped CdTe quantum dots

l ‐glutathione capped highly fluorescent CdTe quantum dots (QDs) were prepared by an aqueous approach and used as fluorescent labels to link albumin bovine serum (BSA) and rat anti‐mouse CD4, which was expressed on mouse T‐lymphocyte and mouse spleen tissue. The sharp and narrow emission peaks showed that the as‐prepared QDs have desirable dispersibility, uniformity and good fluorescence properties. Both CdTe–BSA and CdTe–CD4 conjugates showed an enhancement of fluorescence intensity over that of bare CdTe QDs. The experimental result of gel electrophoresis confirmed the successful conjugation of CdTe–BSA and CdTe–CD4. The fluorescent microscopic images of CdTe–CD4 labeled mouse T‐lymphocyte cells and mouse spleen tissue were compared with that obtained from fluorescein isothiocyanate labeling. It was demonstrated that the CdTe QDs‐based probe exhibited much better photostability and fluorescence intensity than fluorescein isothiocyanate, showing a good application potential in the immuno‐labeling of cells and tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Oligodendroglial and pan‐neural crest expression of Cre recombinase directed by Sox10 enhancer

Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae, and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non‐neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization, and genome manipulation in these populations. genesis 47:765–770, 2009. © 2009 Wiley‐Liss, Inc.     

Generation and characterization of an estrogen receptor alpha‐iCre knock‐in mouse

Two estrogen receptors, ESR1 and ESR2, are responsible for the classical actions of estrogens in mammalian species. They display different spatiotemporal expression patterns and nonoverlapping functions in various tissues and physiological conditions. In this study, a novel knock‐in mouse line that expresses codon‐improved Cre recombinase (iCre) under regulation of the natural Esr1 promoter (Esr1–iCre) was developed. Functional characterization of iCre expression by crossing them with reporter lines (ROSA26‐lacZ or Ai9‐RFP) showed that iCre is faithfully expressed in Esr1‐lineage cells. This novel transgenic mouse line will be a useful animal model for lineage‐tracing Esr1‐expressing cells, selective gene ablation in the Esr1‐lineage cells and for generating global Esr1 knockout mice.     

Neuropilin‐2 genomic elements drive cre recombinase expression in primitive blood,vascular and neuronal lineages

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2‐Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue‐specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709–717, 2015. © 2015 Wiley Periodicals, Inc.