Dynamic characterization of recombinant Chinese hamster ovary cells containing an inducible c-fos promoter GFP expression system as a biomarker.

An inducible reporter gene system for Chinese Hamster Ovary (CHO-DHFR(-)) cells has been developed and characterized with respect to its dynamic properties. The reporter gene system consists of the human c-fos promoter and variants of the green fluorescence protein (GFP), either EGFP with enhanced fluorescence or its destabilized form d2EGFP. The expression of wild-type EGFP or its destabilized form was studied in CHO-DHFR(-) cells in response to serum addition or deprivation. It was shown that serum-induced c-fos promoter mediated EGFP expression was considerably higher than expression from the human CMV promoter, a strong, constitutive promoter preferentially used for high-level expression in CHO cells. However, EGFP was less suitable for studying expression dynamics than d2EGFP due to the protein's long half-life in mammalian cells. The use of d2EGFP resulted in a significant improvement in the dynamic characteristics of the biomarker, particularly when the recombinant cells were selected for high-level GFP expression by subcloning or fluorescence activated cell/sorting (FACS). GFP expression in different subclones and cell populations sorted by FACS was characterized with respect to its dynamic responses in the presence or absence of serum in the culture medium. Significant differences in the GFP expression dynamics were observed for the isolated cell populations. The experimental results indicate that cells with high-level GFP expression also have a faster dynamic response and are thus, desirable for practical application of the reporter gene system e.g. in toxicity monitoring.     

GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。     

Inducible Suppression of Global Translation by Overuse of Rare Codons

Recently, artificial gene networks have been developed in synthetic biology to control gene expression and make organisms as controllable as robots. Here, I present an artificial posttranslational gene-silencing system based on the codon usage bias and low tRNA content corresponding to minor codons. I engineered the green fluorescent protein (GFP) gene to inhibit translation indirectly with the lowest-usage codons to monopolize various minor tRNAs (lgfp). The expression of lgfp interfered nonspecifically with the growth of Escherichia coli, Saccharomyces cerevisiae, human HeLa cervical cancer cells, MCF7 breast cancer cells, and HEK293 kidney cells, as well as phage and adenovirus expansion. Furthermore, insertion of lgfp downstream of a phage response promoter conferred phage resistance on E. coli. Such engineered gene silencers could act as components of biological networks capable of functioning with suitable promoters in E. coli, S. cerevisiae, and human cells to control gene expression. The results presented here show general suppressor artificial genes for live cells and viruses. This robust system provides a gene expression or cell growth control device for artificially synthesized gene networks.     

Targeted gene delivery to mammalian cells by filamentous bacteriophage.

We report that prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the bacteriophage genome. Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor-mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell. As determined by immunofluorescence, F5 phage were efficiently endocytosed into 100 % of ErbB2 expressing SKBR3 cells. To achieve reporter gene expression, F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter. These phage when applied to cells underwent ErbB2-mediated endocytosis leading to GFP expression. GFP expression occurred only in cells overexpressing ErbB2, was dose-dependent reaching, 4 % of cells after 60 hours and was detected with phage titers as low as 2.0 x 10(7) cfu/ml (500 phage/cell). The results demonstrate that bacterial viruses displaying the appropriate antibody can bind to mammalian receptors and utilize the endocytic pathway to infect eukaryotic cells, resulting in expression of a reporter gene inserted into the viral genome. This represents a novel method to discover targeting molecules capable of delivering a gene intracellularly into the correct trafficking pathway for gene expression by directly screening phage antibodies. This should significantly facilitate the identification of appropriate targets and targeting molecules for gene therapy or other applications where delivery into the cytosol is required. This approach can be adapted to directly select, rather than screen, phage antibodies for targeted gene expression. The results also demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle.     

A replication-competent feline leukemia virus, subgroup A (FeLV-A), tagged with green fluorescent protein reporter exhibits in vitro biological properties similar to those of the parental FeLV-A

We previously established that lymphoid tumors could be induced in cats by intradermal injection of ecotropic feline leukemia virus (FeLV), subgroup A, plasmid DNA. In preparation for in vivo experiments to study the cell-to-cell pathway for the spread of the virus from the site of inoculation, the green fluorescent protein (GFP) transgene fused to an internal ribosome entry site (IRES) was inserted after the last nucleotide of the env gene in the ecotropic FeLV-A Rickard (FRA) provirus. The engineered plasmid was transfected into feline fibroblast cells for production of viruses and determination of GFP expression. The virions produced were highly infectious, and the infected cells could continue to mediate strong expression of GFP after long-term propagation in culture. Similar to parental virus, the transgene-containing ecotropic virus demonstrated recombinogenic activity with endogenous FeLV sequences in feline cells to produce polytropic recombinant FeLV subgroup B-like viruses which also contained the IRES-GFP transgene in the majority of recombinants. To date, the engineered virus has been propagated in cell culture for up to 8 months without diminished GFP expression. This is the first report of a replication-competent FeLV vector with high-level and stable expression of a transgene.     

Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.

The introduction of mammalian artificial chromosomes (ACs) into zygotes represents an alternative, more predictive technology for the production of recombinant proteins in transgenic animals. The aim of these experiments was to examine the effects of artificial chromosome microinjection into bovine pronuclei on embryo development and reporter gene expression. Bovine oocytes aspirated from 2-5 mm size follicles were matured in vitro for 22 hr. Mature oocytes were fertilized in vitro with frozen- thawed bull spermatozoa. Artificial chromosome carrying either beta-galactosidase (Lac-Z) gene or green fluorescence protein (GFP) gene were isolated by flow cytometry. A single chromosome was microinjected into one of the two pronuclei of bovine zygotes. Sham injected zygotes served as controls. Injected zygotes were cultured in G 1.2 medium for 7 days. Hatched blastocysts were cultured on blocked STO cell feeder layer for attachment and outgrowth of ICM and trophectoderm cells. The results showed a high zygote survival rate following LacZ-ACs microinjection (74%). However, the blastocyst development rate after 7 days of culture was significantly lower than that of sham injected zygotes (7.5 vs. 22%). Embryonic cells positive for Lac-Z gene were detected by PCR in three of nine outgrowth colonies. In addition, GFP gene expression was observed in 15 out of 85 (18%) embryos at the arrested 2-cell stage to blastocyst stage. Six blastocysts successfully outgrew, three outgrowths were GFP positive for up to 3 weeks in culture. We conclude that the methodology for artificial chromosome delivery into bovine zygotes could lead to viable blastocyst development, and reporter gene expression could be sustained during pre-implantation development.     

Expression of Nkx2-5-GFP bacterial artificial chromosome transgenic mice closely resembles endogenous Nkx2-5 gene activity

Mouse Nkx2-5 gene is essential for early heart development and it is regulated by a complex array of regulatory modules. In order to establish an efficient in vivo system for mapping the Nkx2-5 genomic locus for regulatory regions, we developed improved homologous recombination technology for use in Escherichia coli and then knocked an IRES-hrGFP reporter gene into Nkx2-5 gene in a 120 kb Nkx2-5 bacterial artificial chromosome (BAC) clone. We employed the recombination genes redalpha and redbeta under the pBAD promoter, which was specifically induced by the addition of L-arabinose. Recombination was selected for by our universal targeting cassette which conferred kanamycin resistance in bacterial cells and neomycin resistance in mammalian cells. Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. BAC transgenics using the GFP reporter offers an efficient model system to study gene expression and regulation.     

Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes

Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.     

Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra

Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.     

Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana

The construction of synthetic cell-cell communication networks can improve our quantitative understanding of naturally occurring signaling pathways and enhance our capabilities to engineer coordinated cellular behavior in cell populations. Towards accomplishing these goals in eukaryotes, we developed and analyzed two artificial cell-cell communication systems in yeast. We integrated Arabidopsis thaliana signal synthesis and receptor components with yeast endogenous protein phosphorylation elements and new response promoters. In the first system, engineered yeast 'sender' cells synthesize the plant hormone cytokinin, which diffuses into the environment and activates a hybrid exogenous/endogenous phosphorylation signaling pathway in nearby engineered yeast 'receiver' cells. For the second system, the sender network was integrated into the receivers under positive-feedback regulation, resulting in population density-dependent gene expression (that is, quorum sensing). The combined experimental work and mathematical modeling of the systems presented here can benefit various biotechnology applications for yeast and higher level eukaryotes, including fermentation processes, biomaterial fabrication and tissue engineering.     

Continuous periplasm in a filamentous, heterocyst-forming cyanobacterium

The cyanobacteria bear a Gram-negative type of cell wall that includes a peptidoglycan layer and an outer membrane outside of the cytoplasmic membrane. In filamentous cyanobacteria, the outer membrane appears to be continuous along the filament of cells. In the heterocyst-forming cyanobacteria, two cell types contribute specialized functions for growth: vegetative cells provide reduced carbon to heterocysts, which provide N2-derived fixed nitrogen to vegetative cells. The promoter of the patS gene, which is active specifically in developing proheterocysts and heterocysts of Anabaena sp. PCC 7120, was used to direct the expression of altered versions of the gfp gene. An engineered green fluorescent protein (GFP) that was exported to the periplasm of the proheterocysts through the twin-arginine translocation system was observed also in the periphery of neighbouring vegetative cells. However, if the GFP was anchored to the cytoplasmic membrane, it was observed in the periphery of the producing proheterocysts or heterocysts but not in adjacent vegetative cells. These results show that there is no cytoplasmic membrane continuity between heterocysts and vegetative cells and that the GFP protein can move along the filament in the periplasm, which is functionally continuous and so provides a conduit that can be used for chemical communication between cells.     

小鼠白蛋白启动子的组织特异性功能研究

以绿色荧光蛋白(GFP)基因作为报告基因,通过对比小鼠白蛋白启动子在不同来源细胞系中启动HGFP基因的转录活性,对小鼠白蛋白启动子的组织特异性进行了研究。结果发现,小鼠白蛋白启动子在小鼠肝癌细胞系Hepa 1—6和人肝癌细胞系：HepG2均有很强的转录起始功能,荧光显微镜下可以观察到IGFP表达。Hepa 1—6细胞在转染早期的48h内,CMV的启动子和增强子序列是小鼠白蛋白启动子转录活性的4倍。G418加压筛选2周后,CMV的启动子的转录活性下降到只有小鼠白蛋白启动子活性的1／2。转染人肝癌细胞系HepG2 2周后,荧光显微镜下可以观察到GFP表达。其他的细胞如中华仓鼠卵巢细胞系CHO和人肺癌细胞系PLA 801中转染的小鼠白蛋白启动子不能启动GFP的表达,而对照CMV启动子控制下的GFP基因可在CHO和PLA 801中表达。以上结果说明,小鼠白蛋白启动子仅在肝脏来源的细胞中可以起始下游基因的转录,在其他组织来源的细胞中不能起始转录,这表明小鼠白蛋白启动子具有肝脏组织特异的转录活性,但没有种属特异性。