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.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Tamoxifen‐inducible podocyte‐specific iCre recombinase transgenic mouse provides a simple approach for modulation of podocytes in vivo

We report the generation and initial characterization of a mouse line expressing tamoxifen‐inducible improved Cre (iCre) recombinase (iCre‐ER^T2) under the regulation of NPHS2 (podocin) gene promoter. The resulting transgenic mouse line was named podocin‐iCreER^T2 mice. The efficiency of iCre activity was confirmed by crossing podocin‐iCreER^T2 with the ROSA26 reporter mouse. By using the floxed ROSA reporter mice, we found that tamoxifen specifically induced recombination in the kidneys. In the absence of tamoxifen, recombination was undetectable in podocin‐iCreER^T2;ROSA26 mice. However, following intraperitoneal injection of tamoxifen, selective recombination was observed in the podocytes of adult animals. We further examined the efficiency of recombination by assessing various tamoxifen exposure regimens in adult mice. These results suggest that podocin‐iCre‐ER^T2 mouse provides an excellent genetic tool to examine the function of candidate genes in podocytes in a spatially and temporally‐restricted manner. genesis 48:446–451, 2010. © 2010 Wiley‐Liss, Inc.     

Brain expression of Cre recombinase driven by pancreas‐specific promoters

Cre‐loxP technology enables specific examination of the function and development of individual nuclei in the complex brain network. However, for most brain regions, the utilization of this technique has been hindered by the lack of mouse lines with Cre expression restricted to these regions. Here, we identified brain expressions of three transgenic Cre lines previously thought to be pancreas‐specific. Cre expression driven by the rat‐insulin promoter (Rip‐Cre) was found mainly in the arcuate nucleus, and to a lesser degree in other hypothalamic regions. Cre expression driven by the neurogenin 3 promoter (Ngn3‐Cre mice) was found in the ventromedial hypothalamus. Cre expression driven by the pancreas‐duodenum homeobox 1 promoter (Pdx1‐Cre) was found in several hypothalamic nuclei, the dorsal raphe and inferior olivary nuclei. Interestingly, Pdx1‐Cre mediated deletion of vesicular GABA transporter led to postnatal growth retardation while Ngn3‐Cre mediated deletion had no effects, suggesting a role for Pdx1‐Cre neurons, but not pancreas, in the regulation of postnatal growth. These results demonstrate the potential for these Cre lines to study the function and development of brain neurons. genesis 48:628–634, 2010. © 2010 Wiley‐Liss, 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.     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Cre recombinase‐mediated site‐specific modification of a cellular genome using an integrase‐defective retroviral vector

Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase‐mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild‐type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase‐defective retroviral vectors (IDRV), which contains an ATG‐deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418‐resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site‐specific recombination. The successful substitution of the original viral integration machinery with a non‐viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717–729. © 2010 Wiley Periodicals, Inc.     

Textpresso site‐specific recombinases: A text‐mining server for the recombinase literature including Cre mice and conditional alleles

Textpresso Site Specific Recombinases ( http://ssrc.genetics.uga.edu/ ) is a text‐mining web server for searching a database of more than 9,000 full‐text publications. The papers and abstracts in this database represent a wide range of topics related to site‐specific recombinase (SSR) research tools. Included in the database are most of the papers that report the characterization or use of mouse strains that express Cre recombinase as well as papers that describe or analyze mouse lines that carry conditional (floxed) alleles or SSR‐activated transgenes/knockins. The database also includes reports describing SSR‐based cloning methods such as the Gateway or the Creator systems, papers reporting the development or use of SSR‐based tools in systems such as Drosophila, bacteria, parasites, stem cells, yeast, plants, zebrafish, and Xenopus as well as publications that describe the biochemistry, genetics, or molecular structure of the SSRs themselves. Textpresso Site Specific Recombinases is the only comprehensive text‐mining resource available for the literature describing the biology and technical applications of SSRs. genesis 47:842–846, 2009. © 2009 Wiley‐Liss, Inc.     

Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.     

A unique mouse strain expressing Cre recombinase for tissue-specific analysis of gene function in palate and kidney development

Mammalian palate development is a multistep process, involving initial bilateral downward outgrowth of the palatal shelves from the oral side of the maxillary processes, followed by stage-specific palatal shelf elevation to the horizontal position above the developing tongue and subsequent fusion of the bilateral palatal shelves at the midline to form the intact roof of the oral cavity. While mutations in many genes have been associated with cleft palate pathogenesis, the molecular mechanisms regulating palatal shelf growth, patterning, and elevation are not well understood. Genetic studies of the molecular mechanisms controlling palate development in mutant mouse models are often complicated by early embryonic lethality or gross craniofacial malformation. We report here the development of a mouse strain for tissue-specific analysis of gene function in palate development. We inserted an IresCre bicistronic expression cassette into the 3' untranslated region of the mouse Osr2 gene through gene targeting. We show, upon crossing to the R26R reporter mice, that Cre expression from the Osr2(IresCre) knockin allele activated beta-galactosidase expression specifically throughout the developing palatal mesenchyme from the onset of palatal shelf outgrowth. In addition, the Osr2(IresCre) mice display exclusive Cre-mediated recombination in the glomeruli tissues derived from the metanephric mesenchyme and complete absence of Cre activity in other epithelial and mesenchymal tissues in the developing metanephric kidney. These data indicate that the Osr2(IresCre) knockin mice provide a unique tool for tissue-specific studies of the molecular mechanisms regulating palate and kidney development.     

503unc,a small and muscle‐specific zebrafish promoter

The muscle‐specific UNC‐45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc‐45b upstream region revealed that unc‐45b promoter fragments reliably drive GFP expression after germline transmission. The muscle‐specific 503‐bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(?503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc‐45b expression pattern. We propose that the 503unc promoter is a small and muscle‐specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle. genesis 51:443–447. © 2013 Wiley Periodicals, Inc.     

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.     

Transgenic mice that express Cre recombinase under control of a skeletal muscle-specific promoter from mef2c

Genes expressed in skeletal muscle are often required in other tissues. This is particularly the case for cardiac and smooth muscle, both contractile tissues that share numerous characteristics with skeletal muscle, such that targeted inactivation can lead to embryonic lethality prior to a requirement for gene function in skeletal muscle. Thus, it is essential that conditional inactivation approaches are developed to disrupt genes specifically in skeletal muscle. In this report, we describe a transgenic mouse that expresses Cre recombinase under the control of a skeletal muscle-specific promoter from the mef2c gene. Cre expression in this transgenic line is completely restricted to skeletal muscle from early in development and is present in all skeletal muscles, including those of epaxial and hypaxial origins and in fast and slow fibers. This early skeletal muscle-specific Cre line will be a useful tool to define the function of genes specifically in skeletal muscle.     

A stringent dual control system overseeing transcription and activity of the Cre recombinase for the liver-specific conditional gene knock-out mouse model

Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with several unknown details. However, unlike in other tissues, a tis- sue specific inducible Cre recombinase system that allows temporal and spatial deletion of a target DNA fragment is still not available for in vivo functional gene annotation in hepatocytes. In our pursuit to establish such a mouse model, we designed a dual inducible Cre transgene system and tested it in cultured cells. By combining a CCAAT/enhancer binding protein β (C/EBP β) promoter derived Tet-off expression system and the estrogen receptor (ER) mediated functional control, we show a desirable profile of both hepatocyte-specificity and regulability of the Cre expression in a series of critical assessments in the cell culture system, which provides confidence in continua- tion of our ongoing pursuit in mouse.     

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 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.     

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.     

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.