Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines. Revisions requested 27 October 2005; Revisions received 12 December 2005     

High Level Stable Expression of Pharmacologically Active Human M1–M5 Muscarinic Receptor Subtypes in Mammalian Cells

cDNAs encoding for five mAChR subtypes (M1–M5) were cloned under different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors. Immunofluorescence and flow cytometry revealed that expression of M1–M5 was primarily localized on the cell membrane. Western blotting and radio-ligand binding studies revealed that expression of each receptor was stable at higher passages. These authors contributed equally to this work. Received 22 September 2005; Revisions requested 5 October 2005; Revisions received 2 November 2005; Accepted 4 November 2005     

Rheology and Shear Stress of Centaurea calcitrapa Cell Suspension Cultures Grown in Bioreactor

Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour, when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sⁿ m⁻² run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability loss (90–34%) and phenol accumulation (74–140 μg l⁻¹), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving the possibility to estimate the prevailing broth regime and to optimize bioreactor design. Revisions requested 10 October 2005; Revisions received 19 December 2005     

Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.     

Anti-apoptotic Activity of Laminarin Polysaccharides and their Enzymatically Hydrolyzed Oligosaccharides from Laminaria japonica

Laminarin polysaccharides (LP1) were prepared from Laminaria japonica, a marine brown alga with potential biological activities, by hot water extraction, ultrafiltration and gel chromatography; the molecular weights of the LP1s were between 5 and 10 kDa. Laminarin oligosaccharides (LO) derived by hydrolyzing LP1 with an endo-β-(1→3)-glucanase from Bacillus circulans were mainly di- and penta-oligosaccharides. Treatment of mouse thymocytes with LO or LP1 (1–4 mg ml⁻¹) suppressed apoptotic death around 3- or 2-fold and extended cell survival in culture at a rate of about 30 or 20%. A mouse cDNA microarray showing the genes coding for immune response proteins were induced and apoptotic cell death proteins were reduced significantly by LO provided preliminary information regarding the immunomodulatory mechanism of LO. These results suggest that laminarin oligosaccharides and polysaccharides can be utilized to develop new immunopotentiating substances and functional alternative medicines. Revisions requested 26 October 2005; Revisions received 19 December 2005     

Stem Cell Markers in Gliomas

Gliomas are the most common tumours of the central nervous system (CNS) and a frequent cause of mental impairment and death. Treatment of malignant gliomas is often palliative because of their infiltrating nature and high recurrence. Genetic events that lead to brain tumours are mostly unknown. A growing body of evidence suggests that gliomas may rise from cancer stem cells (CSC) sharing with neural stem cells (NSC) the capacity of cell renewal and multipotency. Accordingly, a population of cells called “side population” (SP), which has been isolated from gliomas on the basis of their ability to extrude fluorescent dyes, behaves as stem cells and is resistant to chemotherapeutic treatments. This review will focus on the expression of the stem cell markers nestin and CD133 in glioma cancer stem cells. In addition, the possible role of Platelet Derived Growth Factor receptor type α (PDGFR-α) and Notch signalling in normal development and tumourigenesis of gliomas are also discussed. Future work elucidating the mechanisms that control normal development will help to identify new cancer stem cell-related genes. The identification of important markers and the elucidation of signalling pathways involved in survival, proliferation and differentiation of CSCs appear to be fundamental for developing an effective therapy of brain tumours. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Bioactivity and pharmacokinetics of two human serum albumin–thymosin α1-fusion proteins, rHSA-Tα1 and rHSA-L-Tα1, expressed in recombinant Pichia pastoris

Thymosin-α1 (Tα1) is indicated for the treatment of certain viral infections, including hepatitis B and C, and cancers, such as melanoma. In this paper, the fusion genes encoding human serum albumin (HSA) and Tα1 with (rHSA-L-Tα1) and without a linker peptide (rHSA-Tα1) were constructed and overexpressed in P. pastoris. Through the process of ion interaction chromatography (Q-Sepharose F.F), hydrophobic interaction chromatography (Phenyl Sepharose HP) and affinity chromatography (Blue Sepharose F.F), the purity of fusion proteins was greater than 97%. In contrast to the reactivity of normal spleen cells to Con A, the data of in vitro murine spleen lymphocytes proliferation experiment suggested that spleen cells achieved a higher degree of T cell maturation after rHSA-L-Tα1, rHSA-Tα1 and Tα1 treatments, respectively. Moreover, rHSA-L-Tα1, rHSA-Tα1 and Tα1 can also antagonize dexamethasone-induced apoptosis of thymocyte sub-populations. In hydrocortisone-induced immunosuppression mice (in vivo experiments), after subcutaneous injections with two fusion proteins and Tα1 for seven consecutive days, the net increment of body weight, the spleen index and the thymus index were significantly improved. Simultaneously, the increase in SOD level and the decrease in MDA level in plasma were observed. The pharmacokinetic data of rHSA-L-Tα1 and rHSA-Tα1 administered in rats showed an improved pharmacokinetic profile with a conspicuous prolonged half life. The analysis of bioactivity and pharmacokinetics suggested that fusion proteins rHSA-L-Tα1 and rHSA-Tα1 were new drug candidates.     

Differential Gene Expression of Human CD34+ Hematopoietic Stem and Progenitor Cells Before and After Culture

Ex vivo expanded CD34⁺ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34⁺ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34⁺ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005     

High-level Expression of an α-l-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the Production of Xylobiose from Xylan

To efficiently produce xylobiose from xylan, high-level expression of an α-l-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an α-l-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine–Dalgarno sequence. A maximum activity of 12 U mg⁻¹ was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed α-l-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and α-l-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 °C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of α-l-arabinofuranosidase gave only half this yield. Revisions requested 27 October 2005; Revisions received 5 September 2005     

Fidelity of a BAC-EGFP transgene in reporting dynamic expression of IL-7Rα in T cells

Interleukin-7 receptor α chain (IL-7Rα)-derived signals are critical for normal T cell development, mature T cell homeostasis, and longevity of memory T cells. IL-7Rα expression in T cells is dynamically regulated at different developmental and antigen-responding stages. However, the molecular mechanism underlying the dynamic regulation is not completely understood. Here we describe generation of a bacterial artificial chromosome (BAC)-based reporter transgenic mouse strain, which contains 210 kb DNA sequence flanking the Il7r locus. We used in vitro validated EGFP reporter and insulator sequences to facilitate the reporter transgene expression. Consistent with endogenous IL-7Rα expression, the BAC transgene was expressed in mature T cells, a portion of natural killer cells but not in mature B cells. In the thymus, the EGFP reporter and endogenous IL-7Rα showed synchronized silencing in CD4⁺CD8⁺ double positive stage, were both upregulated in CD4⁺ or CD8⁺ single positive thymocytes, and both continued to be co-expressed in na?ve T cells in the periphery. Upon encountering antigen, the antigen-specific effector CD8⁺ T cells downregulated both endogenous IL-7Rα and the EGFP reporter, which were upregulated in synchrony in antigen-specific memory CD8 T cells. These results indicate that the BAC-EGFP transgene reports endogenous IL-7Rα regulation with high fidelity, and further suggest that the 210 kb sequence flanking the Il7r locus contains sufficient genetic information to regulate its expression changes in T lineage cells. Our approach thus represents a critical initial step towards systematic dissection of the cis regulatory elements controlling dynamic IL-7Rα regulation during T cell development and cellular immune responses.     

Stromal cells cultured from omentum express pluripotent markers,produce high amounts of VEGF,and engraft to injured sites

When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF–1α, CXCR4, WT–1) and of embryonic pluripotent cells (Oct–4, Nanog, SSEA–1). We have cultured such cells, because they may offer a convenient source of adult stem cells, and have found that they retain stem cell markers and produce high levels of vascular endothelial growth factor for up to ten passages. After systemic or local injection of these cultured cells into rats with acute injury of various organs, the cells specifically engraft at the injured sites. Thus, our experiments show that omental stromal cells can be cultured from activated omentum, and that these cells exhibit stem cell properties enabling them to be used for repair and possibly for the regeneration of damaged tissues.     

The Atlantic Salmon Mhc Class II α and β Promoters are Active in Mammalian Cell Lines

The major histocompatibility complex class II (MHCII) genes are only constitutively expressed in certain immune response cells such as B cells, macrophages, dendritic cells and other antigen presenting cells. This cell specific expression pattern and the presence of conserved regions such as the X-, X2-, Y-, and W-boxes make the MHCII promoters especially interesting as vector constructs. We tested whether the Atlantic salmon (Salmo salar L.) MHCII promoters can function in cell lines from other organisms. We found that the salmon MHCII α and MHCII β promoters could drive expression of a LacZ reporter gene in adherent lymphoblast cell lines from dog (DH82) and rabbit (HybL-L). This paper shows that the promoters of Atlantic salmon MHCII α and β genes can function in mammalian cell lines.     

A Simple and Green Procedure for the Microbial Effective Synthesis of 1-phenylethyl Alcohol in Both Enantiomeric Forms

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40–80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker’s yeast. Revisions requested 29 November 2005; Revisions received 9 January 2006     

Methyl Oleate Modulates <Emphasis Type="Italic">LIP2</Emphasis> Expression in the Lipolytic Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Methyl oleate was used as a primary carbon source and as an alternative inducer for the production of an extracellular lipase, Lip2, in Y. lipolytica strain LgX64.81 grown in a 20-l bioreactor. The lipase-encoding gene, LIP2, was investigated during culture on methyl oleate using a pLIP2–LacZ reporter fusion and we provide evidence for the involvement of methyl oleate in its regulation. Revisions requested 7 July 2005; Revisions received 30 August 2005     

Inducible site-directed recombination in mouse embryonic stem cells.

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly, the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins, tamoxifen can efficiently induce Cre-mediated recombination, thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen, recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein, the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals.     

SK3 Trafficking in Hippocampal Cells: The Role of Different Molecular Domains

The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPΔ578–736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPΔ640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons.     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Redirecting Carbon Flux in Torulopsis glabrata from Pyruvate to α-Ketoglutaric Acid by Changing Metabolic Co-factors

The nutrition conditions needed to redirect the carbon flux in Torulopsis glabrata, a pyruvate hyper-production yeast, from pyruvate to α-ketoglutaric acid (KG) were investigated in a stirred fermentor. A minor amount of KG (1.3 gl⁻¹) was produced when NaOH was used to control the pH, while 12 g KG l⁻¹ was produced when CaCO₃ was used instead. When thiamine and biotin were included in the medium, 13 g KG l⁻¹ and 68 g pyruvate l⁻¹ were produced after 48 h when glucose was nearly consumed (approximately 5 gl⁻¹). With fermentation continuing for a further 16 h, the concentration of pyruvate decreased to 31 gl⁻¹, and KG increased to 30 gl⁻¹. KG thus accumulated at the expense of pyruvate consumption. Received 2 June 2005; Revisions requested 30 June 2005 and 1 September 2005; Revisions received 1 September 2005 and 28 October 2005; Accepted 28 October 2005     

Regulated Expression of a Sindbis Virus Replicon by Herpesvirus Promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and β-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.