SPC-Cre-ERT2 Transgenic Mouse for Temporal Gene Deletion in Alveolar Epithelial Cells

Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER^T2 mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER^T2) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER^T2 activity was first evaluated by crossing SPC-Cre-ER^T2 mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ER^T2 was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER^T2/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER^T2 in a mouse strain bearing TSC1 conditional knockout alleles (TSC1^fx/fx). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER^T2/TSC1^fx/fx mice. Therefore this SPC-Cre-ER^T2 mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.     

Generation of a Tenascin-C-CreER2 Knockin Mouse Line for Conditional DNA Recombination in Renal Medullary Interstitial Cells

Renal medullary interstitial cells (RMIC) are specialized fibroblast-like cells that exert important functions in maintaining body fluid homeostasis and systemic blood pressure. Here, we generated a RMIC specific tenascin-C promoter driven inducible CreER2 knockin mouse line with an EGFP reporter. Similar as endogenous tenascin-C expression, the reporter EGFP expression in the tenascin-C-CreER2^+/− mice was observed in the inner medulla of the kidney, and co-localized with COX2 but not with AQP2 or AQP1, suggesting selective expression in RMICs. After recombination (tenascin-C-CreER2^+/−/ROSA26-lacZ^+/− mice + tamoxifen), β-gal activity was restricted to the cells in the inner medulla of the kidney, and didn''t co-localize with AQP2, consistent with selective Cre recombinase activity in RMICs. Cre activity was not obvious in other major organs or without tamoxifen treatment. This inducible RMIC specific Cre mouse line should therefore provide a novel tool to manipulate genes of interest in RMICs.     

Formation of the Primitive Streak and Mesoderm Cells in Mouse Embryos—Detailed Scanning Electron Microscopical Study

We describe morphological events of the mammalian gastrulation in pre- to middle-primitive-streakstage mouse embryos by using scanning electron microscopy. The first sign of the ingression of the mesodermal cells was disruption of the epithelial structure of ectoderm and the underlying basal lamina, thus forming a semicircular area of the presumptive primitive streak. Then, cells at periphery of the semicircular region spread on the basal lamina by extending many filopodia to it. The majority of the migrating cells formed a loosely arranged cell sheet. We found solitary cells and isolated small groups of cells migrating away from the periphery of the cell sheet. These cells were well spread on the basal lamina, and had large cell processes and many filopodia in the direction of cell migration. Filopodia of these cells were attached to the basal lamina or a meshwork of the extracellular fibrils. These observations suggest that the extracellular matrix serves as the substratum for cell adhesion and migration, and plays an important role in the mammalian gastrulation.     

Intercellular Contacts Between the Embryonic or Extraembryonic Ectoderm and the Primitive Endoderm in Rat Egg Cylinders Prior to the Formation of Primitive Streak

In pre-primitive streak-stage rat egg cylinders, both the embryonic and extraembryonic ectodermal cells projected cytoplasmic protrusions through gaps in the basal lamina and formed intimate cell-to-cell contact with the primitive endodermal cells. The 70 Å microfilaments were considered to participate in the production of these cytoplasmic protrusions. However, direct cell contact mediated by adherent junctions was occasionally found between the embryonic or extraembryonic ectodermal cells and the primitive endodermal cells. It has been proposed that these cell-to-cell contacts may play a role either in the supporting effect of primitive endodermal cells in the maintenance of cellular organization of the ectodermal cells, or in the facilitation of transport of nutritive materials from the primitive endodermal cells to both types of ectodermal cells.     

Transgenic Mouse Line with Green-fluorescent Protein-labeled Centrin 2 allows Visualization of the Centrosome in Living Cells

The centrosome plays diverse roles throughout the cellular mitotic cycle and in post-mitotic cells. Analysis of centrosome position and dynamics in living murine cells has been limited due to a lack of adequate reporters and currently requires either cell fixation/immunostaining or transfection with centrosome reporters. Here we describe the generation and characterization of a transgenic mouse line that constitutively expresses green fluorescent protein-labeled Centrin-2 (GFP-CETN2). The phenotype of the mouse is indistinguishable from wild-type and it displays a single pair of fluorescent centrioles in cells of every organ and time point examined. This model will be helpful for visualizing the centrosome in multiple experimental conditions.     

Enhancement of Interferon Production in a Mouse Cell Line, a High-Yielding Source of Mouse Interferon

Priming with interferon prior to poly(I).poly(C) treatment of a mouse cell line, MO 57/2, followed by sequential administration of metabolic inhibitors, resulted in the production of high yields of mouse interferon.     

Expression of T Protein in the Primitive Streak Is Necessary and Sufficient for Posterior Mesoderm Movement and Somite Differentiation

A characteristic abnormality of chimeras composed of wildtype andT/T(Brachyury) mutant embryonic stem cells is the aggregation and accumulation of mutant cells in the primitive streak and its descendant, the tail bud (V. Wilson, L. Manson, W. C. Skarnes, and R. S. P. Beddington (1995).Development121, 877–886). To demonstrate that this aberrant behaviour of mutant cells in the streak is due only to the absence of wild-type T protein and to investigate dosage effects of T function on cell deployment during gastrulation, a vector expressingTunder the control of its own promoter (which results inTexpression in the primitive streak but not in the notochord) was introduced intoT/Tmutant ES cells carrying a ubiquitouslacZlineage marker. Four clones (TR clones) that express T appropriately in the streak and rescue abnormal chimeric morphology were recovered. In chimeras, these four clones fall into two distinct categories with respect to their ability to exit from the primitive streak and their subsequent tissue colonisation profile. TR1 and TR4 descendants no longer accumulated in the tail bud and gave rise to all types of mesoderm as well as colonising ventral neurectoderm. Interestingly, TR2 and TR5 cells (which express higher levels of T protein than TR1 and TR4in vitro) tended to exit the streak prematurely, showed a marked reduction in posterior mesoderm colonisation, and were virtually excluded from ventral neurectoderm. However, while descendants of all four TR clones can colonise dermomyotome at all axial levels, the parentT/Tmutant cells only contribute to this tissue rostral to the forelimb bud and are completely excluded from more caudal dermomyotome. These results show that the abnormal aggregation of mutant cells homozygous for theBrachyurydeletion (∼200 kb) can be ascribed solely to the lack of wild-type T protein, as can the failure ofT/Tcells to colonise caudal dermomyotome. They also suggest that patterns of cell recruitment from the streak can be influenced by the level of T expression.     

Altered Glycosaminoglycan Chain Structure in a Variant of the C2 Mouse Muscle Cell Line

Abstract: Experiments on the S27 cell line, a variant of the C2 mouse muscle cell line that shows reduced incorporation of ³⁵SO₄ into proteoglycans, suggest that proteoglycans play a role in the clustering of acetylcholine receptors, an early step in synaptogenesis. Thus, unlike the C2 line, S27 myotubes do not form acetylcholine receptor clusters on their surface in aneural cultures and form few clusters in response to agrin. We have examined the proteoglycans synthesized by S27 myotubes to define further the biochemical defect in these cells. Gel filtration analysis of radiolabeled proteoglycans synthesized by C2 and S27 myotubes shows that both cell types express a similarly polydisperse complement of proteoglycans. Both radiolabeled heparan sulfate proteoglycans and chondroitin/dermatan sulfate proteoglycans are reduced in S27 myotubes, with the chondroitin/dermatan sulfate proteoglycans showing a distinct reduction in size. The core protein of perlecan, a major proteoglycan species in muscle, was present in S27 cells and unaltered in electrophoretic mobility. Thus a principal deficiency in S27 cells appears to be a defect in glycosaminoglycan chain elongation.     

Dentition and Jaw of Kokopellia juddi, a Primitive Marsupial or Near-Marsupial from the Medial Cretaceous of Utah

We add to the knowledge of the dentition and lower jaw of the primitive marsupial or near marsupial, Kokopellia juddi, based on newly collected materials from the medial Cretaceous (Albian–Cenomanian) of central Utah. The dental formula, i4 c1 p3 m4, is primitive for (or with respect to) Marsupialia, as are a number of features of the dentary and dentition: presence of a labial mandibular foramen, ?an inflected angle, ?and a trace of the meckelian groove; lack of “staggering” of the lower incisor series; lack of “twinning” between entoconid and hypoconulid on lower molars; incompletely lingual position of lower molar paraconid; upper molar protocone relatively small and mesiodistally narrow; and conules placed about halfway between the protocone and the paracone–metacone. Other than the stylocone, cusps are lacking from the stylar shelf; we argue that this represents the primitive marsupial condition based on the economy of character change and the stratigraphic record of marsupials in the Cretaceous of North America. Recent discoveries of early marsupials, eutherians, and therians of metatherian–eutherian grade provide data indicating that some derived features of the dentary and dentition (e.g., loss of coronoid, meckelian groove, and labial mandibular foramen; acquisition of strong, “winged” conules, double rank postvallum/prevallid shearing, and stylar cusp D) probably arose independently, in some cases more than once, among the major groups of tribosphenic mammals. In turn, this suggests that a common ancestor for marsupials and placentals was more primitive than has generally been appreciated.     

Evaluation of the BDCA2-DTR Transgenic Mouse Model in Chronic and Acute Inflammation

Background and Aims

Plasmacytoid dendritic cells (pDCs) are a small subset of dendritic cells and the main producers of type I interferons. Besides their contribution to tolerance, they are known to be involved in autoimmune diseases and have recently been implicated in atherosclerosis. However, their precise involvement, particularly in advanced lesion development, remains elusive. Hence, we investigated the role of pDCs in atherogenesis vs atheroprogression by specifically depleting this cell population using the BDCA2-DTR mouse model bred to Apolipoprotein E (Apoe ^-/-) deficient mice.

Methods and Results

Our results revealed that continuous diphtheria toxin-induced pDC depletion in Apoe ^-/- BDCA2-DTR mice receiving a high-fat diet (HFD) for 4 weeks did not alter lesion size or composition. Instead, these mice displayed increased B cell numbers and altered levels of inflammatory cytokines. Analysis of depletion efficiency showed that complete pDC depletion could only be sustained for one week and reoccurring pDCs sorted after 4 weeks did not express DTR anymore. Consequently, we analyzed lesion development in a model of partial carotid ligation, inducing established lesions after 5 weeks of HFD feeding, and only depleted pDCs during the last week of 5 weeks HFD feeding. Despite short-term, but efficient pDC depletion, we observed no differences in atherosclerotic lesion development, but changes in inflammatory cytokine titers. To assure the functionality of the BDCA2-DTR model in acute settings, we additionally examined the effect of pDC depletion in an indirect acute lung injury (iALI) model. This time, efficient pDC depletion resulted in a significantly reduced macrophage and neutrophil accumulation in the lung 12 hours after LPS challenge, underlining a pro-inflammatory role of pDCs in the innate immune response in iALI.

Conclusion

Taken together, the BDCA2-DTR mouse model only allows efficient pDC depletion for one week, which subsequently restricts its usability to more acute but not chronic inflammatory disease models.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

Comparative Proteomic Profiling of Dystroglycan-Associated Proteins in Wild Type, mdx, and Galgt2 Transgenic Mouse Skeletal Muscle

Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent-solubilized skeletal muscle. Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycan's roles, both positive and negative, in muscular dystrophy.     

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2^flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2^null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2^null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2^null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.     

In Vitro Selection of High-Infectious, Leukemogenic Virus from Low-Infectious, Non-Leukemogenic Type C Virus from a Malignant ST/a Mouse Cell Line

Low-infectious, nontransforming type C virus was isolated from an in vitro spontaneously transformed ST/a mouse cell line, ST-L1. The virus released by ST-L1 cells was NB-tropic and XC(-). It gave rise to very small peroxidase antibody plaques (PAP) in cultures which initially were nonproducing. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the structural proteins of the ST-L1 virus showed an envelope glycoprotein with an apparent mass of 65 kilodaltons (kdal). The mouse cells SC-1, BALB/3T3, and NIH/3T3 could be productively infected with cell-free supernatants from the ST-L1 cell line; however, virus was detected in supernatant fluids only after two to four subcultures of the infected cells. The virus thus produced was XC(+) and a large plaque former. The virus released from infected SC-1 cells was N-tropic, whereas the viruses from infected NIH/3T3 and BALB/3T3 cells were NB-tropic. The structural proteins of the N- and NB-tropic viruses could be distinguished on SDS polyacrylamide gels, the major dissimilarity being a difference in the mobility of the p30. All these viruses had an envelope glycoprotein with an apparent mass of 70 kdal. The infectivity of the viruses, measured as PAP per nanogram of p30, was 30- to 60-fold lower for the virus released from the ST-L1 cell line than that of the viruses after passage in SC-1, NIH/3T3, and BALB/3T3 cells. None of the viruses could infect rabbit or mink cells. Inoculation of the viruses into newborn mice showed that the ST-L1 virus was non-leukemogenic, whereas the NB-tropic virus selected from this after passage in BALB/3T3 or NIH/3T3 cells was highly leukemogenic. Viruses isolated from leukemic animals were indistinguishable with respect to host range and protein mobilities in SDS gels from the ones with which the mice were inoculated. Although the SC-1-selected virus was highly infectious in vitro, it was only weakly, if at all, leukemogenic.     

hOPG基因启动子驱动报告基因LacZ的转基因小鼠模型的建立

目的:建立带有人类骨保护素OPG基因启动子驱动报告基因LacZ的转基因小鼠模型,为OPG体内转录水平的表达调控研究和药物筛选创造条件。方法:将克隆到的人类OPG基因5′端上游6.0kb非翻译序列作为启动子,大肠杆菌编码β半乳糖苷酶的LacZ基因作为报告基因,构建表达载体pCINeoOPGLacZ。经显微操作注射到受精卵原核中,经PCR以及Southern印迹杂交鉴定转基因阳性小鼠;用RTPCR分析LacZ在组织中的表达;利用邻硝基苯βD半乳吡喃糖苷(ONPG)作为底物反应后比色分析组织中的β半乳糖苷酶活性。结果:构建完成的表达载体pCINeoOPGLacZ质粒经酶切和测序鉴定序列正确,线性化后显微注射。PCR以及Southern印迹杂交鉴定获得了10只转基因小鼠(Founders),经交配繁育,建立了5个转基因小鼠系,RTPCR分析表明其中一个系小鼠组织中表达LacZ基因,与内源OPG表达模式一致,组织中可以广泛检测到相应的β半乳糖苷酶活性。结论:成功建立了人类OPG基因启动子驱动报告基因LacZ的转基因小鼠,为体内研究OPG转录水平的表达及药物筛选提供了理想的动物模型。     

Mitochondrial Dysfunction and Decrease in Body Weight of a Transgenic Knock-in Mouse Model for TDP-43

The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43^A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43^A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.     

A Requirement for FGF Signalling in the Formation of Primitive Streak-Like Intermediates from Primitive Ectoderm in Culture

Background

Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.

Methodology/Principal Findings

Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.

Conclusions/Significance

FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.