Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Smooth muscle α actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full‐length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1–3 copies of the mCherry‐substituted BAC vector. Furthermore, we characterized the expression of SMA‐mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence‐activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA‐expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA‐expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development. genesis 48:457–463, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

STAT5 activation underlies IL7 receptor-dependent B cell development

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R(-/-) background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R(-/-) mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-x(L) expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R(-/-) mice as determined by 1) the restoration of V(H) Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.     

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.     

Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3‐GFP, pOBCol3.6‐GFP, and DMP1‐GFP), similar to the endogenous proteins, are also expressed by bone‐forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP‐Cerulean/DMP1‐Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24‐258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1‐Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP‐Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

miR‐193b availability is antagonized by LncRNA‐SNHG7 for FAIM2‐induced tumour progression in non‐small cell lung cancer

Objectives

Long non‐coding RNAs have identified to involve into the tumour cell proliferation, apoptosis and metastasis. We previously found that up‐regulated LncRNA‐SNHG7 (SNHG7) positively correlated to the Fas apoptosis inhibitory molecule 2 (FAIM2) in lung cancer cells with unclear mechanism.

Methods

Non‐small cell lung cancer (NSCLC) and relative normal tissues (n = 25) were collected. The SNHG7 expression and function in NSCLC was determined. The SNHG7‐miR 193b‐FAIM2 network was analysed in vitro and vivo.

Results

We reported that oncogene SNHG7 predicted a poor clinical outcome and functioned as competitive endogenous RNA (ceRNA) antagonized microRNA‐193b (miR‐193b) to up‐regulate the FAIM2 level in NSCLC. Bioinformatic analysis predicted that SNHG7 harboured miR‐193b‐binding sites, and we found decreased miR‐193b levels in NSCLC tissues when compared to relative normal tissues. Luciferase assays indicated that overexpression of miR‐193b inhibited the Ruc expression of plasmid with miR‐193b‐binding sites of SNHG7 in a dose‐dependent manner. Ectopically expressed SNHG7 also as a molecular sponge sequestered endogenous miR‐193b. Besides, FAIM2 was found to be directly targeted by miR‐193b. The restoration of miR‐193b levels in NSCLC cell lines A549 and H125 suppressed the expression of FAIM2 and related tumour proliferation, metastasis and induced apoptosis. However, forced expression of SNHG7 could down‐regulate miR‐193b to elevate the FAIM2 level of tumour cells, leading to impaired miR‐193b/FAIM2‐induced tumour progression. Knockdown of SNHG7 in vivo significantly delayed the tumour growth with decreased tumour volume, which accompanied with enhanced miR‐193b expression and reduced FAIM2 levels.

Conclusion

The results indicated that miR‐193b is indispensible for the ceRNA role of SNHG7 in FAIM2‐supported tumourigenesis of lung cancer.     

Construction and characterization of a doxycycline‐inducible transgenic system in Msx2 expressing cells

Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2‐rtTA BAC transgenic line which can activate TetO‐Cre expression in Msx2‐expressing cells upon doxycycline (Dox) treatment. Using the Rosa26‐LacZ (R26R) reporter line, we show that rtTA is activated in Msx2‐expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, we show that in body appendages, the transgene can be activated in different domains depending on the timing of Dox treatment. In addition, the transgene can also be effectively activated in adult tissues such as the hair follicle and the urogenital system. Taken together, this Msx2‐rtTA;TetO‐Cre system is a valuable tool for studying gene function in the development of the aforementioned organs in a temporal and spatially‐restricted manner, as well as for tissue lineage tracing of Msx2‐expressing cells. When induced postnatally, this system can also be used to study gene function in adult tissues without compromising normal development and patterning. genesis 47:352–359, 2009. © 2009 Wiley‐Liss, Inc.     

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue‐specific transgene expression in the prostate epithelium has previously been achieved using short prostate‐specific promoters, rendering transgenic mouse lines susceptible to integration site‐dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre‐induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre‐ERT2 protein from small prostate‐specific promoters, these mouse lines will be useful in research focused on prostate‐specific disorders such as benign hyperplasia or cancer. genesis 47:757–764, 2009. © 2009 Wiley‐Liss, Inc.     

A Tie2‐driven BAC‐TRAP transgenic line for in vivo endothelial gene profiling

Recent technological innovations including bacterial artificial chromosome‐based translating ribosome affinity purification (BAC‐TRAP) have greatly facilitated analysis of cell type‐specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC‐TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium‐specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre‐ and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium‐specific mRNAs from brain tissues at different stages. This BAC‐TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. genesis 54:136–145, 2016. © 2016 Wiley Periodicals, Inc.     

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Oligodendroglial and pan‐neural crest expression of Cre recombinase directed by Sox10 enhancer

Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae, and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non‐neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization, and genome manipulation in these populations. genesis 47:765–770, 2009. © 2009 Wiley‐Liss, Inc.     

TPD7 inhibits the growth of cutaneous T cell lymphoma H9 cell through regulating IL‐2R signalling pathway

IL‐2R pathway is a key regulator in the development of immune cells and has emerged as a promising drug target in cancer treatment, but there is a scarcity of related inhibitors. TPD7 is a novel biphenyl urea taspine derivate, which has been shown anti‐cancer effect. Here, we demonstrated the anti‐cancer activity of TPD7 in cutaneous T cell lymphoma and investigated the underlying mechanism of TPD7 through IL‐2R signalling. The inhibitory effect of TPD7 on cell viability exhibited a strong correlation with the expression level of IL‐2R, and cutaneous T cell lymphoma H9 and HUT78 cells were most sensitive to TPD7. TPD7 was nicely bound to IL‐2R and down‐regulated the mRNA and protein levels of IL‐2R. Furthermore, TPD7 suppressed the downstream cascades of IL‐2R including JAK/STAT, PI3K/AKT/mTOR and PLCγ/Raf/MAPK signalling, resulting in Bcl‐2 mitochondrial apoptosis pathway and cell cycle proteins CDK/Cyclins regulation. And, these were verified by flow cytometry analysis that TPD7 facilitated cell apoptosis in H9 cells via mitochondrial pathway and impeded cell cycle progression at G2/M phase. TPD7 is a novel anti‐cancer agent and may be a potential candidate for cutaneous T cell lymphoma treatment by regulating IL‐2R signalling pathway.     

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.     

Highly B lymphocyte‐specific tamoxifen inducible transgene expression of CreERT2 by using the LC‐1 locus BAC vector

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio‐temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte‐specific tamoxifen‐inducible Cre transgenic mouse strain, LC‐1‐hCD19‐CreER^T2. We utilized the human CD19 promoter for expression of the tamoxifen‐inducible Cre recombinase (CreER^T2) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross‐breeding the LC‐1‐hCD19‐CreER^T2 strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC‐1‐hCD19‐CreER^T2 strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse. genesis 47:729–735, 2009. © 2009 Wiley‐Liss, Inc.     

Ceramide plays a prominent role in MDA‐7/IL‐24‐induced cancer‐specific apoptosis

Melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24) uniquely displays broad cancer‐specific apoptosis‐inducing activity through induction of endoplasmic reticulum (ER) stress. We hypothesize that ceramide, a promoter of apoptosis, might contribute to mda‐7/IL‐24 induction of apoptosis. Ad.mda‐7‐infected tumor cells, but not normal cells, showed increased ceramide accumulation. Infection with Ad.mda‐7 induced a marked increase in various ceramides (C16, C24, C24:1) selectively in prostate cancer cells. Inhibiting the enzyme serine palmitoyltransferase (SPT) using the potent SPT inhibitor myriocin (ISP1), impaired mda‐7/IL‐24‐induced apoptosis and ceramide production, suggesting that ceramide formation caused by Ad.mda‐7 occurs through de novo synthesis of ceramide and that ceramide is required for mda‐7/IL‐24‐induced cell death. Fumonisin B1 (FB1) elevated ceramide formation as well as apoptosis induced by Ad.mda‐7, suggesting that ceramide formation may also occur through the salvage pathway. Additionally, Ad.mda‐7 infection enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin in cancer cells. ASMase silencing by RNA interference inhibited the decreased cell viability and ceramide formation after Ad.mda‐7 infection. Ad.mda‐7 activated protein phosphatase 2A (PP2A) and promoted dephosphorylation of the anti‐apoptotic molecule BCL‐2, a downstream ceramide‐mediated pathway of mda‐7/IL‐24 action. Pretreatment of cells with FB1 or ISP‐1 abolished the induction of ER stress markers (BiP/GRP78, GADD153 and pospho‐eIF2α) triggered by Ad.mda‐7 infection indicating that ceramide mediates ER stress induction by Ad.mda‐7. Additionally, recombinant MDA‐7/IL‐24 protein induced cancer‐specific production of ceramide. These studies define ceramide as a key mediator of an ER stress pathway that may underlie mda‐7/IL‐24 induction of cancer‐specific killing. J. Cell. Physiol. 222: 546–555, 2010. © 2009 Wiley‐Liss, Inc.