NG2 cells generate both oligodendrocytes and gray matter astrocytes

NG2 glia constitute a fourth major glial cell type in the mammalian central nervous system (CNS) that is distinct from other cell types. Although circumstantial evidence suggests that some NG2 glia differentiate into oligodendrocytes, their in vivo fate has not been directly examined. We have used the bacterial artificial chromosome (BAC) modification technique to generate transgenic mice that express DsRed or Cre specifically in NG2-expressing (NG2+) cells. In NG2DsRedBAC transgenic mice, DsRed was expressed specifically in NG2+ cells throughout the postnatal CNS. When the differentiation potential of NG2+ cells in vitro was examined using DsRed+NG2+ cells purified from perinatal transgenic brains, the majority of the cells either remained as NG2+ cells or differentiated into oligodendrocytes. In addition, DsRed+NG2+ cells also differentiated into astrocytes. The in vivo fate of NG2 glia was examined in mice that were double transgenic for NG2creBAC and the Cre reporter Z/EG. In the double transgenic mice, the Cre reporter EGFP was detected in myelinating oligodendrocytes and in a subpopulation of protoplasmic astrocytes in the gray matter of ventrolateral forebrain but not in fibrous astrocytes of white matter. These observations suggest that NG2+ cells are precursors of oligodendrocytes and some protoplasmic astrocytes in gray matter.     

Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis. In addition, we demonstrate that at least some of the Ccr4p/Pop2p-associated Not proteins are cytoplasmic, and lesions in some of the NOT genes can lead to defects in mRNA deadenylation rates. The Ccr4p deadenylase is inhibited in vitro by addition of the poly(A) binding protein (Pab1p), suggesting that dissociation of Pab1p from the poly(A) tail may be rate limiting for deadenylation in vivo. In addition, the rapid deadenylation of the COX17 mRNA, which is controlled by a member of the Pumilio family of deadenylation activators Puf3p, requires an active Ccr4p/Pop2p/Not deadenylase. These results define the Ccr4p/Pop2p/Not complex as the cytoplasmic deadenylase in yeast and identify positive and negative regulators of this enzyme complex.     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector

Generation of mammalian cells stably expressing multiple exogenous genes is currently difficult. Here we provide a strategy to facilitate this process. First, a helper vector p2A containing three coding sequences for viral 2A peptides was constructed. Three reporter genes coding for red fluorescent protein (DsRed), firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) were then inserted into p2A to form a fusion open reading frame that was subsequently subcloned into a lentiviral vector. After transduction, EGFP-positive 293T cells were selected by fluorescence activated cell sorting. The expression of exogenous genes in selected cells was stable for more than 15 passages, and EGFP-positive cells were over 95%. The efficient cleavages of 2A-peptide mediated polyprotein were also observed and all three reporter proteins were functional. Thus, a stable DsRed/Fluc/EGFP-coexpressing cell line was readily established within a short time. The strategy could be useful for basic research and protein production. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a novel transgenic mouse line expressing Cre recombinase under the control of the Cdx2 neural specific enhancer

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal‐related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R‐YFP reporter mice have indicated that the Cdx2NSE‐Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double‐transgenic embryos has revealed that this novel Cre‐driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso‐ventral axis (including pre‐migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non‐cranial neural crest derivatives of double‐transgenic embryos. Therefore, we believe that the Cdx2NSE‐Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 51:777–784. © 2013 Wiley Periodicals, Inc.     

FlEx‐based transgenic reporter lines for visualization of Cre and Flp activity in live zebrafish

Site‐specific recombinases such as Cre and Flp are invaluable tools for genetic manipulations, but their usage in zebrafish has been limited. Incorporating recently developed flip‐excision (FlEx) design that allows stable inversions, we have established zebrafish reporter lines that express bright and ubiquitous EGFP, but switch to express mCherry in the presence of Cre or Flp. Here, we demonstrate the stable inversion in the reporter lines, both in somatic cells and in the germ line by Cre or Flp, and the subsequent reinversion using the other recombinase. Using the reporter lines, we characterized cardiomyocyte‐specific Cre lines and neuronal progenitor‐specific and tamoxifen‐dependent Cre lines. We also used the reporter lines for screening Cre‐ and Flp‐based enhancer trap lines. Similar to the widely used Cre reporter lines in mice, these FlEx‐based reporter lines will facilitate the use of recombinases for genetic manipulations in zebrafish. genesis 47:484–491, 2009. © 2009 Wiley‐Liss, Inc.     

Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease

Microglia are the principal immune cells of the brain. In Alzheimer disease, these brain mononuclear phagocytes are recruited from the blood and accumulate in senile plaques. However, the role of microglia in Alzheimer disease has not been resolved. Microglia may be neuroprotective by phagocytosing amyloid-beta (Abeta), but their activation and the secretion of neurotoxins may also cause neurodegeneration. Ccr2 is a chemokine receptor expressed on microglia, which mediates the accumulation of mononuclear phagocytes at sites of inflammation. Here we show that Ccr2 deficiency accelerates early disease progression and markedly impairs microglial accumulation in a transgenic mouse model of Alzheimer disease (Tg2576). Alzheimer disease mice deficient in Ccr2 accumulated Abeta earlier and died prematurely, in a manner that correlated with Ccr2 gene dosage, indicating that absence of early microglial accumulation leads to decreased Abeta clearance and increased mortality. Thus, Ccr2-dependent microglial accumulation plays a protective role in the early stages of Alzheimer disease by promoting Abeta clearance.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter

Background

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene''s native ATG start site has not been widely available.

Methodology

Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.

Conclusions

Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.     

Generation of a mouse line expressing Cre recombinase in glycinergic interneurons

In caudal regions of the CNS, glycine constitutes the major inhibitory neurotransmitter. Here, we describe a mouse line that expresses Cre recombinase under the control of a BAC transgenic glycine transporter 2 (GlyT2) promoter fragment. Mating of GlyT2‐Cre mice with the Cre reporter mouse lines Rosa26/LacZ and Rosa26/YFP and analysis of double transgenic offsprings revealed strong transgene activity in caudal regions of the central nervous system, i.e., brain stem and spinal cord. Some additional Cre expression was observed in cortical and cerebellar regions. In brain stem and spinal cord, Cre expressing cells were identified as glycinergic interneurons by staining with GlyT2‐ and glycine‐immunoreactive antibodies; here, >80% of the glycine‐immunoreactive cells expressed the Cre reporter protein. These data indicate that GlyT2‐Cre mice are a useful tool for the genetic manipulation of glycinergic interneurons. genesis 48:437–445, 2010. © 2010 Wiley‐Liss, Inc.     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electropo-rated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targetin     

A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action

Cre-mediated site-specific recombination allows conditional transgene expression or gene knockouts in mice. Inducible Cre recombination systems have been developed to bypass initial embryonic lethal phenotypes and provide access to later embryonic or adult phenotypes. We have produced Cre transgenic mice in which excision is tamoxifen inducible and occurs in a widespread mosaic pattern. We utilized our Cre excision reporter system combined with an embryonic stem (ES) cell screen to identify ES cell clones with undetectable background Cre activity in the absence of tamoxifen but efficient excision upon addition of tamoxifen. The CreER transgenic mouse lines derived from the ES cells were tested using the Z/AP and Z/EG Cre reporter lines. Reporter gene expression indicated Cre excision was maximal in midgestation embryos by 2 days after tamoxifen administration, with an overall efficiency of 5-10% of cells with Cre excision. At 3 days after tamoxifen treatment most reporter gene expression marked groups of cells, suggesting an expansion of cells with Cre excision, and the proportion of cells with Cre excision was maintained. In adults, Cre excision was also observed with varying efficiencies in all tissues after tamoxifen treatment.