A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

A series of Cre‐ERT2 drivers for manipulation of the skeletal muscle lineage

We report the generation of five mouse strains with the tamoxifen‐inducible Cre (Cre‐ER^T2; CE) gene cassette knocked into the endogenous loci of Pax3, Myod1, Myog, Myf6, and Myl1, collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R^LacZ, during embryogenesis and show that they direct tightly controlled tamoxifen‐inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen‐inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock‐in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. Although we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology. genesis 52:759–770, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of Nppa‐tagBFP reporter knock‐in mouse line for studying cardiac chamber specification

Nppa is a cardiac hormone which plays critical roles in regulating salt–water balance. Its expression is restricted to the atria of the healthy post‐natal heart. During heart development, spatio‐temporal expression of Nppa is dynamically changed within the heart and becomes restricted to the atria upon birth. In contrast to its atrial specific expression after birth, Nppa is re‐expressed in the adult ventricles in response to cardiac hypertrophy. To study cardiac chamber specification during development and pathological cardiac remodeling during heart disease, we generated a novel Nppa reporter mouse line by knocking‐in a tagBFP reporter cassette into 3′‐UTR of the Nppa gene without disrupting the endogenous gene. Our results demonstrated dynamic tagBFP expression in the developing heart, recapitulating the spatiotemporal expression pattern of endogenous Nppa. We also found that Nppa‐tagBFP is induced in the ventricle during pathological remodeling. Taken together, Nppa‐tagBFP reporter knock‐in mouse model described in this article will serve as a valuable tool to study cardiac chamber specification during development as well as pathological cardiac remodeling.     

Tamoxifen‐inducible Cre‐mediated recombination in adipocytes

To generate a mouse line which allows inducible, Cre/loxP‐dependent recombination in adipocytes, we used RedE/RedT‐mediated recombineering to insert the CreER^T2‐transgene, which encodes a fusion protein of Cre and a mutated tamoxifen‐responsive estrogen receptor, into the start codon of the adipocyte‐specific Adipoq gene. Adipoq encodes adiponectin, an adipokine specifically expressed in differentiated adipocytes. Tamoxifen treatment induced almost complete recombination in white adipose tissue of the AdipoqCreER^T2 mouse line (97%–99%), while no recombination was seen in vehicle‐treated animals. Recombination in brown adipose tissue was about 15%, whereas other organs and tissues did not undergo recombination. In addition, mice expressing CreER^T2 in adipocytes did not show any alterations of metabolic functions like glucose tolerance, lipolysis, or energy expenditure compared to control mice. Therefore the AdipoqCreER^T2 mouse line will be a valuable tool for studying the consequences of a temporally controlled deletion of floxed genes in white adipose tissue. genesis 48:618–625, 2010. © 2010 Wiley‐Liss, Inc.     

Musashi1‐CreERT2: A new cre line for conditional mutagenesis in neural stem cells

The RNA‐binding protein Musashi1 (Msi1) is one of two mammalian homologues of DrosophilaMusashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long‐term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1‐expressing cells have self‐renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1‐CreER^T2 knock‐in mice and show by cell lineage tracing that Msi1‐CreER^T2‐expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1‐CreER^T2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development. genesis, 51:128–134, 2013. © 2012 Wiley Periodicals, Inc.     

A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant‐negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER^T2 mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER^T2 mouse is a valuable tool for in vivo time‐dependent and region‐specific knockout and overexpression studies. genesis 48:207–212, 2010. © 2010 Wiley‐Liss, Inc.     

Creation of zebrafish knock‐in reporter lines in the nefma gene by Cas9‐mediated homologous recombination

CRISPR/Cas9‐based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)‐induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock‐ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9‐targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock‐in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.     

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ER^T2). In the background of the Cre reporter mouse strain Rosa26^lacZ, we show that the fusion CreER^T2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 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.     

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.