Yeast Ypt11 is targeted to recycling endosomes in mammalian cells

BACKGROUND INFORMATION: In yeast, Ypt11 or Ypt32 along with the highly homologous Ypt8 or Ypt31 has been reported to be an essential component of intra-Golgi trafficking and has been implicated in the budding of vesicles from the most distal Golgi compartment. RESULTS AND CONCLUSIONS: In the present study, we show that, in human cells, after heterologous expression of GFP-Ypt11 (where GFP stands for green fluorescent protein), the protein is targeted to transferrin-positive recycling endosomes. This compartment has been shown to form extensive tubular networks on applying the drug Brefeldin A. We also show, by confocal fluorescent microscopy, that these networks also contain Rab11 in cells expressing CFP-Rab11a (where CFP stands for cyan fluorescent protein) fusion protein and that these structures are identical with those targeted by GFP-Ypt11.     

表达绿色荧光蛋白突变体的重组伪狂犬病毒的构建及其增殖特性

将绿色荧光蛋白突变体M 1(EGFPS14 7/P)基因融合到伪狂犬病毒 (PRV)非必需糖蛋白 gG的第 8个氨基酸下游 ,通过同源重组、空斑纯化和PCR筛选获得能表达M 1并导致gG基因部分缺失的重组病毒 gG-/M1 。重组病毒经Southern杂交、Western印迹和荧光观察证实构建正确。纯化的重组病毒以低感染指数接种PK 15细胞 ,在感染早期 (6h)就能观察到荧光 ,随着病毒的增殖 ,荧光逐渐增强 (2 4～ 36h) ,直至完全病变 ,荧光淬灭。进一步对重组病毒gG-/M1 与亲本株gG-/LacZ 、野毒株的增殖特性进行比较 ,发现 3种毒株在增殖滴度上无显著差异。上述结果表明构建的PRVgG-/M1 突变株能作为活细胞示踪实时监测病毒感染的动态分析。     

ELP_(30)-tag蛋白纯化能力的原核表达研究

目的:探究一种小分子量类弹性蛋白标签(elastin-like protein tag,ELP tag)——ELP_(30)-tag在原核表达系统中的蛋白纯化能力。方法:人工合成ELP_(30)-tag基因并将其构建于pET-28a(+)载体,结合2种内含肽(intein1和intein2)基因和增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)基因,构建4个含有不同元件序列的原核表达载体:pET-ELP_(30)、pET-ELP_(30)-eGFP、pET-ELP_(30)-intein1-eGFP和pET-eGFP-intein2-ELP_(30);将表达载体转化大肠杆菌BL21(DE3),经IPTG诱导重组蛋白表达,并通过可逆相变循环(inverse transition cycling,ITC)纯化重组蛋白ELP_(30)、ELP_(30)-eGFP、ELP_(30)-intein1-eGFP和eGFP-intein2-ELP_(30),随后通过调节溶液pH值或添加二硫苏糖醇(DL-Dithiothreitol,DTT)分别诱导intein1和intein2断裂,最后再经ITC分离获得纯eGFP。结果:利用设计的ELP_(30)-tag成功纯化获得了重组蛋白ELP_(30)、ELP_(30)-eGFP和eGFP-intein2-ELP_(30);重组蛋白ELP_(30)-intein1-eGFP和eGFP-intein2-ELP_(30)中的内含肽可经诱导发生断裂而释放eGFP,但未能分离获得纯eGFP。由此为小分子量ELP-tag的运用和优化设计奠定了一定基础。     

Retrovirus-mediated gene transfer and expression of EGFP in chicken

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.     

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

Zinc‐finger nucleases (ZFNs) are engineered nucleases that induce DNA double‐strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non‐homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non‐homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP ‐ZFN mRNAs into the embryos of an EGFP ‐transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP ‐ZFNs at off‐target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on‐target site with less effect on the off‐target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.     

低分子肝素的绿色荧光蛋白荧光标记及其活性检测

增强型绿色荧光蛋白（EGFP）是生物领域常用的标记物. 本实验首先利用高碘酸法活化低分子肝素（LMWH）,与EGFP连接得到LMWH EGFP.然后用Sephacryl S-200 HR对其进行初步分离,再用Sephadex G-10 HR进行脱盐纯化. 采用Sephacryl S 200 HR检测LMWH EGFP的纯度,为单一对称峰. 经检测,LMWH-EGFP具有良好的热稳定性和耐碱性. 通过荧光分光检测器检测LMWH-EGFP的λEx为488 nm,λEm为509 nm. 通过抗凝实验发现LMWH EGFP仍具有抗凝活性. 本实验建立了LWMH荧光标记的方法,为多糖的荧光标记提供了新的选择.     

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNγ) were treated using short interfering RNA (siRNA) and short‐hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNγ was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094–1105. © 2009 Wiley Periodicals, Inc.     

Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat     

Application of Cell-Surface Engineering for Visualization of Yeast in Bread Dough: Development of a Fluorescent Bio-Imaging Technique in the Mixing Process of Dough

Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at ?30 °C and sliced at 10 μm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.     

Dynamics of DNA replication factories in living cells

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.     

Low-temperature pausing of cultivated mammalian cells

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.     

Use of internally nuclease‐protected single‐strand DNA oligonucleotides and silencing of the mismatch repair protein,MSH2, enhances the replication of corrected cells following gene editing

Background

Gene editing is potentially a powerful technology for introducing genetic changes by using short single‐stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S‐ or G2‐phase. In the present study, we evaluate whether a novel ssODN design and down‐regulation of MSH2 expression allows the isolation of replicating gene‐edited cells.

Methods

Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction events by flow cytometry. Converted cells were isolated by cell sorting and grown to determine colony formation efficiencies. MSH2 expression was suppressed with small interfering RNA and the cell cycle distribution of cells transfected with ssODN was quantified by flow cytometry, following propidium iodide or DRAQ5 staining.

Results

Although efficiency was higher using ssODN end‐protected with phosphorothioate, the potential of edited cells to form colonies was lower than those targeted with unmodified ssODN. We established that ssODN transfection itself perturbs the cell cycle and that MSH2 gene silencing increases correction efficiency. In both cases, however, the effect was dependent on the positioning of the protected nucleotides. Importantly, when internally protected ssODN was used in combination with MSH2 suppression, a higher proportion of G1‐phase corrected cells was observed 48–64 h after transfection.

Conclusions

Use of internally protected ssODN and downregulating cellular MSH2 activity may facilitate isolation of viable, actively replicating gene‐edited cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor

Tagged G‐protein‐coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N‐terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C‐terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N‐terminus, or EGFP located at the C‐terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 56–68, 2002     

巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP.利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株.用EGFP标记菌接种小麦'小偃107'种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况.结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦'小偃107'植株在根系发育、株高、分蘖数等方面比对照有较明显的优势.研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用.     

Cytoplasmic foci are sites of mRNA decay in human cells

Understanding gene expression control requires defining the molecular and cellular basis of mRNA turnover. We have previously shown that the human decapping factors hDcp2 and hDcp1a are concentrated in specific cytoplasmic structures. Here, we show that hCcr4, hDcp1b, hLsm, and rck/p54 proteins related to 5'-3' mRNA decay also localize to these structures, whereas DcpS, which is involved in cap nucleotide catabolism, is nuclear. Functional analysis using fluorescence resonance energy transfer revealed that hDcp1a and hDcp2 interact in vivo in these structures that were shown to differ from the previously described stress granules. Our data indicate that these new structures are dynamic, as they disappear when mRNA breakdown is abolished by treatment with inhibitors. Accumulation of poly(A)(+) RNA in these structures, after RNAi-mediated inactivation of the Xrn1 exonuclease, demonstrates that they represent active mRNA decay sites. The occurrence of 5'-3' mRNA decay in specific subcellular locations in human cells suggests that the cytoplasm of eukaryotic cells may be more organized than previously anticipated.     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

Mesenchymal stem cells: Paracrine signaling and differentiation during cutaneous wound repair

Cutaneous wounds persist as a health care crisis in spite of increased understanding of the cellular and molecular responses to injury. Contributing significantly to this crisis is the lack of reliable therapies for treatment of wounds that are slow to heal including chronic wounds and deep dermal wounds that develop hypertrophic scars. This article will review the growing evidence demonstrating the promise of multipotent mesenchymal stem/stromal (MSCs) for the treatment of impaired wound healing. MSCs are often referred to as mesenchymal stem cells despite concerns that these cells are not truly stem cells given the lack of evidence demonstrating self-renewal in vivo. Regardless, abundant evidence demonstrates the therapeutic potential of MSCs for repair and regeneration of damaged tissue due to injury or disease. To date, MSC treatment of acute and chronic wounds results in accelerated wound closure with increased epithelialization, granulation tissue formation and angiogenesis. Although there is evidence for MSC differentiation in the wound, most of the therapeutic effects are likely due to MSCs releasing soluble factors that regulate local cellular responses to cutaneous injury. Important challenges need to be overcome before MSCs can be used effectively to treat wounds that are slow to heal.     

显微注射法制备转基因家蝇技术的建立

旨在利用显微注射法对早期家蝇(Musca domestica L.)卵注射含有增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的转座子载体,实现活体基因稳定表达并对其进行验证,为开展家蝇基因功能的研究奠定基础.文中自制适用于显微注射家蝇卵的硼硅酸盐玻璃微量注射针,...     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997