Conditional expression of Lodavin, an avidin-tagged LDL receptor, for biotin-mediated applications in vivo

Lodavin represents an engineered fusion protein that consists of a cytoplasmic and a transmembrane domain of the human low‐density lipoprotein receptor coupled to an extracellular avidin monomer. Biotinylated compounds have been successfully targeted to Lodavin‐expressing cells that have been transduced by a Lodavin‐containing virus, and the targeting is based on the high affinity between biotin and avidin. We engineered a Rosa26 (R26R) knock‐in Lodavin mouse to develop biotin‐based applications such as targeted drug delivery, cell purification, and tissue imaging in vivo. A cDNA encoding Lodavin was inserted downstream of a floxed βgeo resistance gene in the R26R locus in embryonic stem cells, and a germ line‐derived R26RLodavin mouse line was generated. Efficient removal of the floxed βgeo cassette and conditional activation of Lodavin expression was achieved as a result of crossing the R26RLodavin mice with HoxB7‐Cre, Wnt4‐Cre, or Tie1‐Cre mice. In summary, the R26RLodavin mouse line may provide a useful tool for testing and developing applications with the aid of avidin and biotin interaction. genesis 50:693–699, 2012. © 2012 Wiley Periodicals, Inc.     

hCD2-iCre and Vav-iCre Mediated Gene Recombination Patterns in Murine Hematopoietic Cells

Cre-recombinase mediated conditional deletion of Lox-P site flanked ("floxed") genes is widely used for functional gene annotation in mice. Many different Cre-transgenic mouse lines have been developed for cell-type specific gene disruption. But often, the precise tissue-patterns of Cre activity remain incompletely characterized. Two widely used transgenes for conditional gene recombination in hematopoietic cells are Vav-iCre driven from the murine Vav1 promotor, and hCD2-iCre driven from the human CD2 promotor. Vav-iCre expresses active Cre in fetal and adult hematopoietic stem cells and all descendants, hCD2-iCre in immature and mature B and T lymphocytes. To better characterize which hematopoietic cells contain hCD2-iCre activity, we compared EYFP fluorescence in hCD2-iCre^+/- R26-stop-EYFP^+/- and Vav-iCre^+/- R26-stop-EYFP^+/-mice. R26-stop-EYFP ubiquitously encodes EYFP preceded by a floxed stop cassette. By removing it, Cre activity induces measurable EYFP expression. Our results confirm the known activity patterns for both Cre transgenes and unveil additional hCD2-iCre mediated reporter gene recombination in common lymphoid progenitors, in natural killer cells and their progenitors, and in plasmacytoid and conventional dendritic cells. This supports previously proposed common lymphoid origins for natural killer cells and subsets of dendritic cells, and indicates the need to consider pleiotropic effects when studying hCD2-iCre mediated conditional knockout mice. Vav-iCre^+/- R26-stop-EYFP^+/-mice did not show the non-hematopoietic recombination in vascular endothelial cells seen in other Vav-Cre mouse lines, but displayed an unexpected Vav-iCre mediated recombination in a bone cell subset lacking hematopoietic markers. This pinpoints the need to consider stromal cell contributions to phenotypes of Vav-iCre mediated conditional knockout mice. Altogether, our data provide the first detailed assessment of hCD2-iCre and Vav-iCre mediated deletion of floxed genes during lymphocyte development from hematopoietic stem cells and open up novel applications for either Cre-transgenic mouse line.     

Pdgfrb‐Cre targets lymphatic endothelial cells of both venous and non‐venous origins

The Pdgfrb‐Cre line has been used as a tool to specifically target pericytes and vascular smooth muscle cells. Recent studies showed additional targeting of cardiac and mesenteric lymphatic endothelial cells (LECs) by the Pdgfrb‐Cre transgene. In the heart, this was suggested to provide evidence for a previously unknown nonvenous source of LECs originating from yolk sac (YS) hemogenic endothelium (HemEC). Here we show that Pdgfrb‐Cre does not, however, target YS HemEC or YS‐derived erythro‐myeloid progenitors (EMPs). Instead, a high proportion of ECs in embryonic blood vessels of multiple organs, as well as venous‐derived LECs were targeted. Assessment of temporal Cre activity using the R26‐mTmG double reporter suggested recent occurrence of Pdgfrb‐Cre recombination in both blood and lymphatic ECs. It thus cannot be excluded that Pdgfrb‐Cre mediated targeting of LECs is due to de novo expression of the Pdgfrb‐Cre transgene or their previously established venous endothelial origin. Importantly, Pdgfrb‐Cre targeting of LECs does not provide evidence for YS HemEC origin of the lymphatic vasculature. Our results highlight the need for careful interpretation of lineage tracing using constitutive Cre lines that cannot discriminate active from historical expression. The early vascular targeting by the Pdgfrb‐Cre also warrants consideration for its use in studies of mural cells. genesis 54:350–358, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, 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.     

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.     

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.     

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.     

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.     

A new tool for conditional gene manipulation in a subset of keratin‐expressing epithelia

Megsin is a serine protease inhibitor (Serpin) that has known expression in kidney mesangial cells. Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Megsin regulatory elements. When crossed to the ROSA26R‐lacZ reporter mice, the Megsin‐Cre transgene mediates loxP recombination primarily in the skin, forestomach, and esophagus, but surprisingly not in the mesangial cells. Within the skin, cells in all epidermal layers and the hair follicle cells expressed Cre. This transgene also has uniform expression in the epithelium of the forestomach and esophagus. Conditional deletion of Adam10, a gene known to have important functions in skin development, by using this Megsin‐Cre transgene led to severe skin defects. In addition, these mutants appear to have reduced folds and surface area in the forestomach. These results show that the Megsin‐Cre transgene can mediate loxP‐recombination in all epidermal layers of the skin, the hair follicle cells, as well as in the epithelium of the forestomach and esophagus, all of which have known expression of various keratins. This Megsin‐Cre transgene can serve as a new tool for conditional genetic manipulation to study development and diseases in the skin and the upper digestive tract. genesis 50:899–907, 2012. © 2012 Wiley Periodicals, Inc.     

Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease

The I–CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double‐strand breaks and enhancing DNA recombination reactions in maize cells. The DNA‐binding properties of this protein were re‐designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single‐chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium‐mediated transformation, the cleavage resulted in mutations at a co‐delivered extra‐chromosomal ms26‐site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26‐site in 5.8% of transgenic T₀ plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double‐strand break repair generated by non‐homologous end joining. One of 21 mutagenized T₀ plants carried two mutated alleles of the MS26 gene. As expected, the bi‐allelic mutant T₀ plant and the T₁ progeny homozygous for the ms26 mutant alleles were male‐sterile. This paper described the second maize chromosomal locus (liguless‐1 being the first one) mutagenized by a re‐designed I–CreI–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.     

IL7‐hCD25 and IL7‐Cre BAC transgenic mouse lines: New tools for analysis of IL‐7 expressing cells

IL‐7 is a cytokine that is required for T‐cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL‐7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL‐7 synthesis, specific cell types involved and molecular mechanisms regulating IL‐7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL‐7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL‐7.hCD25 BAC transgene, detected by reactivity with anti‐hCD25 antibody, mimicked endogenous IL‐7 expression. Fetal and adult tissues from crosses between IL‐7.Cre transgenic mice and Rosa26R or R26‐EYFP reporters demonstrated X‐gal or YFP staining in tissues known to express endogenous IL‐7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL‐7 producing cells in various tissues and to manipulate gene expression selectively in IL‐7 expressing cells. genesis 47:281–287, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

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.