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 Hoxa13:Cre mouse strain for conditional gene manipulation in developing limb,hindgut, and urogenital system

The developing limb is a useful model for studying organogenesis and developmental processes. Although Cre alleles exist for conditional loss‐ or gain‐of‐function in limbs, Cre alleles targeting specific limb subdomains are desirable. Here we report on the generation of the Hoxa13:Cre line, in which the Cre gene is inserted in the endogenous Hoxa13 gene. We provide evidence that the Cre is active in embryonic tissues/regions where the endogenous Hoxa13 gene is expressed. Our results show that cells expressing Hoxa13 in developing limb buds contribute to the entire autopod (hand/feet) skeleton and validate Hoxa13 as a distal limb marker as far as the skeleton is concerned. In contrast, in the limb musculature, Cre‐based fate mapping shows that almost all muscle masses of the zeugopod (forearm) and part of the triceps contain Hoxa13‐expressing cells and/or their descendants. Besides the limb, the activity of the Cre is detectable in the urogenital system and the hindgut, primarily in the epithelium and smooth muscles. Together our data show that the Hoxa13:Cre allele is a useful tool for conditional gene manipulation in the urogenital system, posterior digestive tract, autopod and part of the limb musculature. genesis 53:366–376, 2015. © 2015 Wiley Periodicals, Inc.     

Off‐target assessment of CRISPR‐Cas9 guiding RNAs in human iPS and mouse ES cells

The CRISPR‐Cas9 system consists of a site‐specific, targetable DNA nuclease that holds great potential in gene editing and genome‐wide screening applications. To apply the CRISPR‐Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off‐target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ～90 predicted off‐target sites per gRNA. In a Cas9‐based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on‐target sites and in only a few off‐target sites genome‐wide in human 293FT, human‐induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on‐target and off‐target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off‐target activity. Our results show that off‐target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off‐target site. Off‐target Cas9 activity can be minimized by selecting gRNAs with few off‐target sites of near complementarity. genesis 53:225–236, 2015. © 2014 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Generation and characterization of a Ddx4-iCre transgenic line for deletion in the germline beginning at genital ridge colonization

Germline-specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole-body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved Cre (iCre) sequence in-frame at the 3′ end of the Ddx4 locus to generate the Ddx4-P2A-iCre allele. Our functional assessment of this new allele, designated Ddx4^iCreJoBo, reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5–12.5) germline deletion when compared to the inducible Oct4^CreERT2 line. We found the Ddx4^iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26R^LacZ and R26R^tdTomato) and a floxed gene of interest (Cripto^flox) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for Ddx4^iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4^iCreJoBo line as useful for germline studies in which early gonadal deletion is required.     

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.     

The power of zebrafish models for understanding the co‐occurrence of craniofacial and limb disorders

Craniofacial and limb defects are two of the most common congenital anomalies in the general population. Interestingly, these defects are not mutually exclusive. Many patients with craniofacial phenotypes, such as orofacial clefting and craniosynostosis, also present with limb defects, including polydactyly, syndactyly, brachydactyly, or ectrodactyly. The gene regulatory networks governing craniofacial and limb development initially seem distinct from one another, and yet these birth defects frequently occur together. Both developmental processes are highly conserved among vertebrates, and zebrafish have emerged as an advantageous model due to their high fecundity, relative ease of genetic manipulation, and transparency during development. Here we summarize studies that have used zebrafish models to study human syndromes that present with both craniofacial and limb phenotypes. We discuss the highly conserved processes of craniofacial and limb/fin development and describe recent zebrafish studies that have explored the function of genes associated with human syndromes with phenotypes in both structures. We attempt to identify commonalities between the two to help explain why craniofacial and limb anomalies often occur together.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

Biased sex‐ratio and sex‐biased heterozygote disadvantage affect the maintenance of a genetic polymorphism and the properties of hybrid zones

The evolution of biodiversity is a major issue of modern biology, and it is becoming increasingly topical as the ongoing erosion of diversity puts serious threats on human well‐being. An elementary mechanism that allows maintaining diversity is the interplay between dispersal and heterozygote selective disadvantage, which can lead to self‐sustainable spatial genetic structures and is central to the stability of hybrid zones. Theoretical studies supporting the importance of this mechanism assume a balanced sex‐ratio and a heterozygote disadvantage equally affecting both sexes, despite the multiplicity of empirical evidence that (i) adult sex‐ratio is usually biased towards either male or female and that (ii) heterozygote disadvantage often affects a single sex. We expanded the existing theory by weighting the strength of selection against heterozygote according to the biased in sex‐ratio and in heterozygote disadvantage. The range of conditions allowing for the maintenance of polymorphism can then either double or vanish. We discuss the implications of such finding for birds, mammals and insects diversity. Finally, we provide simple analytical predictions about the effect of those biased on the width and speed of hybrid zones and on the time for the spread of beneficial mutations through such zones.     

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non‐syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three “deleter” Cre strains: Gdf9‐Cre, CAG‐Cre, and Ella‐Cre. When Cre expression was driven by the Gdf9‐Cre or CAG‐Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella‐Cre transgenic line resulted in incomplete recombination, despite expression at the one‐cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre‐driver used.     

A conditional allele of Rspo3 reveals redundant function of R‐spondins during mouse limb development

Summary: R‐spondins are secreted ligands that bind cell surface receptors and activate Wnt/β‐catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1‐4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R‐spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R‐spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue‐specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.     

Generation and characterization of a conditional deletion allele for Lmna in mice

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4–8 postnatal (P) weeks or P16–18 days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna^flox/flox mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna^Δ/Δ) generated by germline Cre expression display postnatal lethality at P16–18 days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins.     

Generation of mice carrying a knockout‐first and conditional‐ready allele of transforming growth factor beta2 gene

Transforming growth factor beta2 (TGFβ2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular, and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFβ2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell‐matrix remodeling, epithelial‐mesenchymal transition, and wound healing in a highly context‐dependent and tissue‐specific manner. Tgfb2^?/? mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2^βgeo (knockout‐first lacZ‐tagged insertion) gene‐trap allele and Tgfb2^flox conditional allele. Tgfb2^βgeo/βgeo or Tgfb2^βgeo/‐ mice died at perinatal stage from the same congenital heart defects as Tgfb2^?/? mice. β‐galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2^βgeo fetal tissue sections. Tgfb2^flox mice were produced by crossing the Tgfb2^+/βgeo mice with the FLPeR mice. Tgfb2^flox/? mice were viable. Tgfb2 conditional knockout (Tgfb2^cko/?) fetuses were generated by crossing of Tgfb2^flox/? mice with Tgfb2^+/?; EIIaCre mice. Systemic Tgfb2^cko/? embryos developed cardiac defects which resembled the Tgfb2^βgeo/βgeo, Tgfb2^βgeo/?, and Tgfb2^?/? fetuses. In conclusion, Tgfb2^βgeo and Tgfb2^flox mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFβ2 in embryonic development, adult organs, and disease pathogenesis and cancer. genesis 52:817–826, 2014. © 2014 Wiley Periodicals, Inc.     

Generation and characterization of a mouse line carrying Reck‐CreERT2 knock‐in allele

Reck encodes a membrane‐anchored glycoprotein implicated in the regulation of extracellular metalloproteinases, Notch‐signaling, and Wnt7‐signaling and shown to play critical roles in embryogenesis and tumor suppression. Precise mechanisms of its actions in vivo, however, remain largely unknown. By homologous recombination, we generated a new Reck allele, Reck^CreERT2 (MGI symbol: Reck<tm3.1(cre/ERT2)Noda>). This allele is defective in terms of Reck function but expected to induce loxP‐mediated recombination in the cells committed to express Reck. Similarity in the expression patterns of the Reck^CreERT2 transgene and the endogenous Reck gene was confirmed in five tissues. In the adult hippocampus, induction of Reck expression after transient cerebral ischemia could be demonstrated using this allele. These results indicate the utility of this Cre‐driver allele in further studies.     

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.     

Reproductive strategies and isolation‐by‐demography in a marine clonal plant along an eutrophication gradient

Genetic diversity in clonal organisms includes two distinct components, (i) the diversity of genotypes or clones (i.e. genotypic richness) in a population and (ii) that of the alleles (i.e. allelic and gene diversity within populations, and differentiation between populations). We investigated how population differentiation and genotypic components are associated across a gradient of eutrophication in a clonal marine plant. To that end, we combined direct measurements of sexual allocation (i.e. flower and seed counts) and genotypic analyses, which are used as an estimator of effective sexual reproduction across multiple generations. Genetic differentiation across sites was also modelled according to a hypothesis here defined as isolation‐by‐demography, in which we use population‐specific factors, genotypic richness and eutrophication that are hypothesized to affect the source‐sink dynamics and thus influence the genetic differentiation between a pair of populations. Eutrophic populations exhibited lower genotypic richness, in agreement with lower direct measurements of sexual allocation and contemporaneous gene flow. Genetic differentiation, while not explained by distance, was best predicted by genotypic richness and habitat quality. A multiple regression model using these two predictors was considered the best model (R² = 0.43). In this study, the relationship between environment and effective sexual–asexual balance is not simply (linearly) predicted by direct measurements of sexual allocation. Our results indicate that population‐specific factors and the isolation‐by‐demography model should be used more often to understand genetic differentiation.     

Development and characterization of novel tetra‐ and dinucleotide microsatellite markers for the French Polynesia black‐lipped pearl oyster,Pinctada margaritifera

Ten microsatellite loci were developed for the black‐lipped pearl oyster Pinctada margaritifera with a magnetic bead enrichment protocol. These tetra‐ and dinucleotide markers were polymorphic, with 10 to 43 alleles observed in 97 individuals from two Tuamotu atoll populations. Most loci revealed significant genic differentiation between the two populations and also exhibited some degree of heterozygote deficiencies, probably due to the presence of null alleles. These loci should be very useful to describe genetic structure, genetic variability and reproductive success in the various aquaculture and wild populations of pearl oyster in French Polynesia.