Generation of a KOR‐Cre knockin mouse strain to study cells involved in kappa opioid signaling

The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non‐neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR‐Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR‐Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR‐Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR‐Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR‐Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR. genesis 54:29–37, 2016. © 2015 Wiley Periodicals, Inc.     

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.     

Generation of BAF53b‐Cre transgenic mice with pan‐neuronal Cre activities

Molecular and functional studies of genes in neurons in mouse models require neuron‐specific Cre lines. The current available neuronal Cre transgenic or knock‐in lines either result in expression in a subset of neurons or expression in both neuronal and non‐neuronal tissues. Previously we identified BAF53b as a neuron‐specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b‐Cre is largely neuron‐specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b‐Cre functioned in primary cultures in a pan‐neuron‐specific manner. Thus, BAF53b‐Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies. genesis, 53:440–448, 2015. © 2015 Wiley Periodicals, Inc.     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

Generation of a NK1R‐CreER knockin mouse strain to study cells involved in Neurokinin 1 Receptor signaling

The Neurokinin 1 Receptor (NK1R), which binds Substance P, is expressed in discrete populations of neurons throughout the nervous system, where it has numerous roles including the modulation of pain and affective behaviors. Here, we report the generation of a NK1R‐CreER knockin allele, in which CreER^T2 replaces the coding sequence of the TACR1 gene (encoding NK1R) in order to gain genetic access to these cells. We find that the NK1R‐CreER allele mediates recombination in many regions of the nervous system that are important in pain and anxiety including the amygdala, hypothalamus, frontal cortex, raphe nucleus, and dorsal horn of the spinal cord. Other cell types that are labeled by this allele include amacrine cells in the retina and fibroblasts in the skin. Thus, the NK1R‐CreER mouse line is a valuable new tool for conditional gene manipulation enabling the visualization and manipulation of cells that express NK1R.     

Nrl‐Cre transgenic mouse mediates loxP recombination in developing rod photoreceptors

The developing mouse retina is a tractable model for studying neurogenesis and differentiation. Although transgenic Cre mouse lines exist to mediate conditional genetic manipulations in developing mouse retinas, none of them act specifically in early developing rods. For conditional genetic manipulations of developing retinas, a Nrl‐Cre mouse line in which the Nrl promoter drives expression of Cre in rod precursors was created. The results showed that Nrl‐Cre expression was specific to the retina where it drives rod‐specific recombination with a temporal pattern similar to endogenous Nrl expression during retinal development. This Nrl‐Cre transgene does not negatively impact retinal structure and function. Taken together, the data suggested that the Nrl‐Cre mouse line was a valuable tool to drive Cre‐mediated recombination specifically in developing rods. genesis 54:129–135, 2016. © 2016 Wiley Periodicals, Inc.     

Generation of a MOR‐CreER knock‐in mouse line to study cells and neural circuits involved in mu opioid receptor signaling

Mu opioid receptor (MOR) is involved in various brain functions, such as pain modulation, reward processing, and addictive behaviors, and mediates the main pharmacologic effects of morphine and other opioid compounds. To gain genetic access to MOR‐expressing cells, and to study physiological and pathological roles of MOR signaling, we generated a MOR‐CreER knock‐in mouse line, in which the stop codon of the Oprm1 gene was replaced by a DNA fragment encoding a T2A peptide and tamoxifen (Tm)‐inducible Cre recombinase. We show that the MOR‐CreER allele undergoes Tm‐dependent recombination in a discrete subtype of neurons that express MOR in the adult nervous system, including the olfactory bulb, cerebral cortex, striosome compartments in the striatum, hippocampus, amygdala, thalamus, hypothalamus, interpeduncular nucleus, superior and inferior colliculi, periaqueductal gray, parabrachial nuclei, cochlear nucleus, raphe nuclei, pontine and medullary reticular formation, ambiguus nucleus, solitary nucleus, spinal cord, and dorsal root ganglia. The MOR‐CreER mouse line combined with a Cre‐dependent adeno‐associated virus vector enables robust gene manipulation in the MOR‐enriched striosomes. Furthermore, Tm treatment during prenatal development effectively induces Cre‐mediated recombination. Thus, the MOR‐CreER mouse is a powerful tool to study MOR‐expressing cells with conditional gene manipulation in developing and mature neural tissues.     

Generation of Elf5‐Cre knockin mouse strain for trophoblast‐specific gene manipulation

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5‐Cre mice, we mated Elf5‐Cre mice with Rosa26^mT/mG reporter mice, and found that Elf5‐Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5‐Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5‐Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.     

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.     

Inducible cardiomyocyte‐specific gene disruption directed by the rat Tnnt2 promoter in the mouse

We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by coinjection of two transgenes, a “reverse” tetracycline‐controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline‐responsive promoter (TetO). Here, Tnnt2‐rtTA activated TetO‐Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte‐specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2‐rtTA;TetO‐Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods. genesis 48:63–72, 2010. © 2009 Wiley‐Liss, Inc.     

Generation of Smad7‐Cre recombinase mice: A useful tool for the study of epithelial‐mesenchymal transformation within the embryonic heart

Smad7 can be induced by various transforming growth factor‐β superfamily ligands and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. Previous analyses have demonstrated that although Smad7 is widely expressed, it is predominantly found in the vascular endothelium. Because of the restricted spatiotemporal reporter expression driven via a novel 4.3 kb Smad7 promoter in endocardial cells overlying the hearts atrioventricular (AV) cushions; we hypothesized that a transgenic Cre line would prove useful for the analysis of endocardial cushion and valve formation. Here we describe a mouse line, Smad7^Cre, where Cre is robustly expressed within both cardiac outflow and AV endocardial cushions. Additionally, as endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme, we crossed the Smad7^Cre mice to the ROSA26^eGFP‐DTA diphtheria toxin A‐expressing mice in order to genetically ablate Smad7^Cre expressing cells. Ablation of Smad7^Cre cells resulted in embryonic lethality by E11.5 and largely acellular endocardial cushions. genesis 47:469–475, 2009. © 2009 Wiley‐Liss, Inc.     

NB‐3 signaling mediates the cross‐talk between post‐traumatic spinal axons and scar‐forming cells

Little is known about the molecules mediating the cross‐talk between post‐traumatic axons and scar‐forming cells after spinal cord injury. We found that a sustained NB‐3 induction was simultaneously present in the terminations of post‐traumatic corticospinal axons and scar‐forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced in vivo with NB‐3 deficiency or interruption of NB‐3 trans‐homophilic interactions. Biochemical, in vitro and in vivo evidence demonstrated that NB‐3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar‐forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB‐3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB‐3 trans‐homophilic interactions mediate the cross‐talk between post‐traumatic axons and scar‐forming cells and impair the intrinsic growth ability of injured axons.     

An X‐linked Myh11‐CreERT2 mouse line resulting from Y to X chromosome‐translocation of the Cre allele

The Myh11‐CreER^T2 mouse line (Cre⁺) has gained increasing application because of its high lineage specificity relative to other Cre drivers targeting smooth muscle cells (SMCs). This Cre allele, however, was initially inserted into the Y chromosome (X/Y^Cre+), which excluded its application in female mice. Our group established a Cre⁺ colony from male ancestors. Surprisingly, genotype screening identified female carriers that stably transmitted the Cre allele to the following generations. Crossbreeding experiments revealed a pattern of X‐linked inheritance for the transgene (k > 1000), indicating that these female carries acquired the Cre allele through a mechanism of Y to X chromosome translocation. Further characterization demonstrated that in hemizygous X/X^Cre+ mice Cre activity was restricted to a subset arterial SMCs, with Cre expression in arteries decreased by 50% compared to X/Y^Cre+ mice. This mosaicism, however, diminished in homozygous X^Cre+/X^Cre+ mice. In a model of aortic aneurysm induced by a SMC‐specific Tgfbr1 deletion, the homozygous X^Cre+/X^Cre+ Cre driver unmasked the aortic phenotype that is otherwise subclinical when driven by the hemizygous X/X^Cre+ Cre line. In conclusion, the Cre allele carried by this female mouse line is located on the X chromosome and subjected to X‐inactivation. The homozygous X^Cre+/X^Cre+ mice produce uniform Cre activity in arterial SMCs.     

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.     

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.