Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Long‐term repopulation of aged bone marrow stem cells using young Sca‐1 cells promotes aged heart rejuvenation

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca‐1) cells to reconstitute aged BM and rejuvenate the aged heart, and examined the underlying molecular mechanisms. BM Sca‐1⁺ or Sca‐1^? cells from young (2–3 months) or aged (18–19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca‐1⁺, young Sca‐1^?, old Sca‐1⁺, and old Sca‐1^?. Four months later, expression of rejuvenation‐related genes (Bmi1, Cbx8, PNUTS, Sirt1, Sirt2, Sirt6) and proteins (CDK2, CDK4) was increased along with telomerase activity and telomerase‐related protein (DNA‐PKcs, TRF‐2) expression, whereas expression of senescence‐related genes (p16^INK4a, P19^ARF, p27^Kip1) and proteins (p16^INK4a, p27^Kip1) was decreased in Sca‐1⁺ chimeric hearts, especially in the young group. Host cardiac endothelial cells (GFP^?CD31⁺) but not cardiomyocytes were the primary cell type rejuvenated by young Sca‐1⁺ cells as shown by improved proliferation, migration, and tubular formation abilities. C‐X‐C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP⁺) cells isolated from young Sca‐1⁺ chimeric hearts. Protein expression of Cxcr4, phospho‐Akt, and phospho‐FoxO3a in endothelial cells derived from the aged chimeric heart was increased, especially in the young Sca‐1⁺ group. Reconstitution of aged BM with young Sca‐1⁺ cells resulted in effective homing of functional stem cells in the aged heart. These young, regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells.     

Generation of Pecam1 endothelial specific dual reporter mouse model

Endothelial cells are specialized epithelium lining the interior surface of vessels and play fundamental roles in angiogenesis, vascular permeability, and immune response. To identify endothelial cells in vivo, we constructed a Pecam1^{nlacZ‐H2B‐GFP/+} knock‐in mouse model in which the endothelial cells are labeled by nuclear LacZ (nlacZ) expression. When Pecam1^{nlacZ‐H2B‐GFP/+} mice are bred with germline Cre deleter mice, Pecam1^H2B‐GFP/+ line is created with native nuclear GFP (H2B‐GFP) expression in the endothelium of various organs. This dual reporter mouse provides us with a powerful genetic tool for definitive identification of endothelial cells and monitoring this important cell population throughout development, homeostasis, and disease conditions in mammals.     

Bmi‐1 high‐expressing cells enrich cardiac stem/progenitor cells and respond to heart injury

Bmi‐1 gene is well recognized as an oncogene, but has been recently demonstrated to play a role in the self‐renewal of tissue‐specific stem cells. By using Bmi‐1^GFP/+ mice, we investigated the role of Bmi‐1 in cardiac stem/progenitor cells and myocardial repair. RT‐PCR and flow cytometry analysis indicated that the expression of Bmi‐1 was significantly higher in cardiac side population than the main population from CD45^?Ter119^?CD31^? heart cells. More Sca‐1⁺ cardiac stem/progenitor cells were found in Bmi‐1 GFP^hi subpopulation, and these Bmi‐1 GFP^hi heart cells showed the potential of differentiation into SMM⁺ smooth muscle‐like cells and TnT⁺ cardiomyocyte‐like cells in vitro. The silencing of Bmi‐1 significantly inhibited the proliferation and differentiation of heart cells. Otherwise, myocardial infarction induced a significantly increase (2.7‐folds) of Bmi‐1 GFP^hi population, mainly within the infarction and border zones. These preliminary data suggest that Bmi‐1^hi heart cells are enriched in cardiac stem/progenitor cells and may play a role in myocardial repair.     

Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis‐resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response‐1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na⁺/Pi) cotransporter PiT‐1. Since the effects of an inhibitor of Na⁺/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2α and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi‐induced phosphorylation of ERK1/2 in the cells where the expression of PiT‐1 was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT‐1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

The fibroblast growth factor‐2 is not essential for melanoma formation in a transgenic mouse model

Fibroblast growth factor 2 (FGF2) has been assigned a role in melanocyte proliferation and in development of human cutaneous melanoma. We have used a transgenic mouse melanoma model in combination with mice lacking mouse FGF2 to analyse the possible implication of FGF2 in melanomagenesis. Tyr::N‐ras^Q61K transgenic mice which are deficient for FGF2 and the tumor suppressors p16^INK4a and p19^ARF are hyperpigmented and develop cutaneous metastasizing melanoma, with no difference to mice wildtype for FGF2. We conclude from our data, that FGF2 is not essential for melanoma progression and metastasis.     

RUSH and CRUSH: A rapid and conditional RNA interference method in mice

RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26^lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G₀ transgenic founders by tetraploid complementation. A control CRUSH^GFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSH^GFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.     

Pluripotency and Chimera Competence of an Embryonic Stem Cell Line from the Sea Perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

A stable GFP-expressing (GFP⁺LJES1) cell strain was developed from the LJES1 cells obtained from sea perch (Lateolabrax japonicus,) embryos. GFP⁺LJES1 cells were induced in vitro by RA to differentiate into a variety of cell types and also had the ability to form embryoid body-like structures in suspension culture. To determine the differentiation potential of LJES1 cells in vivo, GFP⁺LJES1 cells were transplanted into sea perch and zebrafish embryos at mid-blastula stage. Twenty out of 478 transplanted sea perch embryos contained GFP-expressing LJES1 cells 24 h after microinjection. Fifteen chimera embryos developed into fry. In these chimeras, the GFP⁺LJES1 cells contributed to a variety of tissues including the head and trunk. In zebrafish, 221 embryos were microinjected with GFP⁺LJES1 cells and 22 chimera embryos and fries expressing GFP were obtained. Donor GFP⁺LJES1 cells contributed to various tissues in head and trunk of zebrafish embryos and hatched fry.     

HPC‐1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival

Two types of syntaxin 1 isoforms, HPC‐1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A^?/? mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B^?/? mice using targeted gene disruption and investigated their phenotypes. STX1B^?/? mice were born alive, but died before postnatal day 14, unlike STX1A^?/? mice. Morphologically, brain development in STX1B^?/? mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B^?/? neurons was lower than that of WT or STX1A^?/? neurons after 9 days. Interestingly, STX1B^?/? neurons survived on WT or STX1A^?/? glial feeder layers as well as WT neurons. However, STX1B^?/? glial feeder layers were less effective at promoting survival of STX1B^?/? neurons. Conditioned medium from WT or STX1A^?/? glial cells had a similar effect on survival, but that from STX1B^?/? did not promote survival. Furthermore, brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 supported survival of STX1B^?/? neurons. BDNF localization in STX1B^?/? glial cells was disrupted, and BDNF secretion from STX1B^?/? glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia.

     

Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin

Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5^Cre-driven GFP expression, we identified in addition to the expected GFP⁺ myogenic cells (presumably satellite cells), a second dominant GFP⁺ population distinguished as being Sca1⁺, non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5^Cre-driven reporter when compared to the MyoD^Cre driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5^Cre-driven GFP⁺ cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP⁺ cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5^Cre-driven GFP⁺ cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.     

The peripheral chimerism of bone marrow–derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice

Bone marrow–derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP⁺) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP⁺lin⁻Sca-1⁺ cells into non-haematopoietic tissues. The transplantation of BM cells or GFP⁺lin⁻Sca-1⁺ cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP⁺ cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP⁺lin⁻Sca-1⁺ cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.     

Fgf3‐Fgf4‐cis: A new mouse line for studying Fgf functions during mouse development

The fibroblast growth factor (FGF) family consists of 22 ligands in mice and humans. FGF signaling is vital for embryogenesis and, when dysregulated, can cause disease. Loss‐of‐function genetic analysis in the mouse has been crucial for understanding FGF function. Such analysis has revealed that multiple Fgfs sometimes function redundantly. Exploring such redundancy between Fgf3 and Fgf4 is currently impossible because both genes are located on chromosome 7, about 18.5 kb apart, making the frequency of interallelic cross‐over between existing mutant alleles too infrequent to be practicable. Therefore, we retargeted Fgf3 and Fgf4 in cis, generating an Fgf3 null allele and a conditional Fgf4 allele, subject to Cre inactivation. To increase the frequency of cis targeting, we used an F1 embryonic stem cell line that contained 129/SvJae (129) and C57BL/6J (B6) chromosomes and targeting constructs isogenic to the 129 chromosome. We confirmed cis targeting by assaying for B6/129 allele‐specific single‐nucleotide polymorphisms. We demonstrated the utility of the Fgf3^Δ‐Fgf4^flox‐cis mouse line by showing that the caudal axis extension defects found in the Fgf3 mutants worsen when Fgf4 is also inactivated. This Fgf3^Δ‐Fgf4^flox‐cis line will be useful to study redundancy of these genes in a variety of tissues and stages in development. genesis 54:91–98, 2016. Published 2016. This article is a US Government work and is in the public domain in the USA.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

A mouse model for visualization of GABAB receptors

GABA_B receptors are the G‐protein‐coupled receptors for the neurotransmitter γ‐aminobutyric acid (GABA). Receptor subtypes are based on the subunit isoforms GABA_B1a and GABA_B1b, which combine with GABA_B2 subunits to form heteromeric receptors. Here, we used a modified bacterial artificial chromosome (BAC) containing the GABA_B1 gene to generate transgenic mice expressing GABA_B1a and GABA_B1b subunits fused to the enhanced green fluorescence protein (eGFP). We demonstrate that the GABA_B1‐eGFP fusion proteins reproduce the cellular expression patterns of endogenous GABA_B1 proteins in the brain and in peripheral tissue. Crossing the GABA_B1‐eGFP BAC transgene into the GABA_B1^?/? background restores pre and postsynaptic GABA_B functions, showing that the GABA_B1‐eGFP fusion proteins substitute for the lack of endogenous GABA_B1 proteins. Finally, we demonstrate that the GABA_B1‐eGFP fusion proteins replicate the temporal expression patterns of native GABA_B receptors in cultured neurons. These transgenic mice therefore provide a validated tool for direct visualization of native GABA_B receptors. genesis 47:595–602, 2009. © 2009 Wiley‐Liss, Inc.     

Systemic administration of bone marrow‐derived cells leads to better uterine engraftment than use of uterine‐derived cells or local injection

Stem cells are recruited to the uterus where they differentiate into endometrial cells and have been suggested as potential therapy for uterine injury such as Asherman's syndrome. However, it is unknown whether local intrauterine injection may result in better stem cell engraftment of the uterus compared with systemic administration, and whether uterine‐derived cells (UDCs) may confer an advantage over BM‐derived cells (BMDCs). Mice underwent local injury to a single uterine horn. Green fluorescent protein (GFP)‐expressing BMDCs, UDCs or saline (control) were injected either intravenously or locally (uterine lumen) into wild‐type recipients. Two or 3 weeks post‐transplant, uterine tissues were collected for fluorescence‐activated cell sorting (FACS) and immunohistochemistry/immunofluorescence studies. Mice injected intravenously with BMDCs or UDCs had increased GFP⁺ cells recruitment to the non‐injured or injured uterus compared to those injected locally. No significant differences were noted in GFP⁺ cell recruitment to the injured versus non‐injured horn. In addition, systemic injection of BMDCs led to greater recruitment of GFP⁺ cells at 2 weeks and 3 weeks compared with UDCs. Immunohistochemical staining demonstrated that GFP⁺ cells were found in stroma but not in epithelium or blood vessels. Immunofluorescence analysis revealed that GFP⁺ cells were mostly CD45‐negative, and negative for CD31 and cytokeratin, confirming their stromal identity. In conclusion, the systemic route of administration results in better recruitment of BMDCs or UDCs to the injured uterus than local injection. In addition, BMDCs recruitment to the uterus is greater than UDCs. These findings inform the development of stem cell‐based therapies targeting the uterus.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP⁺ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP⁺ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.