eGFP Fusion Gene Research Articles

Construction of Setosphaeria turcica STK1-EGFP fusion gene vector and its expression in Pichia pastoris

STK1 is one important MAPK gene regulating the conidial development, osmotic stress and pathogenicity of Setosphaeria turcica. At first, the Pichia pastoris GS115 expression vector pPIC3.5K-EGFP containing enhanced green fluorescent protein gene (EGFP) was constructed, then STK1 gene was first amplified by PCR with the template of cDNA of S. turcica model isolate 01-23, and then cloned into the vector pPIC3.5K-EGFP with enhanced green fluorescent protein gene (EGFP) to construct the STK1-EGFP fusion gene expression vector pPIC3.5K-STK1-EGFP. The vector was transformed into the susceptible cells of Pichia pastoris GS115 by electric shock process, and the transformants were identified by MD medium screening and PCR determination. The STK1 gene and EGFP gene could be expressed effectively and stably in the transformants as detected by RT-PCR and fluorescence observation. In addition, we also found that the Kozak sequence before the start codon of STK1 gene could increase 4.8 folds expression level of STK1- EGFP fusion gene. The above research results laid a good foundation for subcellular localization and antibody preparation of STK1 protein.

Comparison of the induction of c-fos-eGFP and Fos protein in the rat spinal cord and hypothalamus resulting from subcutaneous capsaicin or formalin injection

We evaluated whether a c-fos-enhanced green fluorescent protein (eGFP) transgenic rat line, which expresses the c-fos and eGFP fusion gene, can be useful for the study of nociceptive pathways and processing. Capsaicin solution (15%) or formalin (5%) was subcutaneously injected bilaterally into the hind paws (100μL per each paw) of adult male c-fos-eGFP transgenic or wild-type rats. Control rats were injected with ethanol or physiological saline respectively. Transgenic and wild-type rats were perfused at 1.5, 3 and 6h post injection, with some transgenic rats being perfused 24h post injection. A comparison of eGFP in transgenic rats and Fos-like immunoreactivity (LI) in wild-type rats was made in the dorsal spinal cord, paraventricular nucleus (PVN) and supraoptic nucleus (SON). Oxytocin-LI (OXT-LI) was carried out to examine the activation of OXT neurons in the PVN and SON. Following capsaicin or formalin treatment, eGFP was maximally expressed at 6h in the spinal cord and 3h in the PVN and SON, whereas, Fos-LI was maximally expressed at 1.5h in all the regions we analyzed. Induction of eGFP in the OXT neurons was observed after capsaicin or formalin treatment, while Fos-LI in the OXT neurons was observed only after formalin treatment. These results demonstrate that the peak induction of c-fos-eGFP following exposure to acute nociceptive stimuli was delayed by around 1.5–4.5h, but more sensitive than endogenous Fos, suggesting that the c-fos-eGFP rat line can be useful for the study of nociceptive pathways and processing.

2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori.

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.

A cotton LIM domain-containing protein (GhWLIM5) is involved in bundling actin filaments

LIM-domain proteins play important roles in cellular processes in eukaryotes. In this study, a LIM protein gene, GhWLIM5, was identified in cotton. Quantitative RT-PCR analysis showed that GhWLIM5 was expressed widely in different cotton tissues and had a peak in expression during fiber elongation. GFP fluorescence assay revealed that cotton cells expressing GhWLIM5:eGFP fusion gene displayed a network distribution of eGFP fluorescence, suggesting that GhWLIM5 protein is mainly localized to the cell cytoskeleton. When GhWLIM5:eGFP transformed cells were stained with rhodamine-phalloidin there was consistent overlap in eGFP and rhodamine-palloidin signals, demonstrating that GhWLIM5 protein is colocalized with the F-actin cytoskeleton. In addition, high-speed cosedimentation assay verified that GhWLIM5 directly bound actin filaments, while low cosedimentation assay and microscopic observation indicated that GhWLIM5 bundled F-actin in vitro. Increasing amounts of GhWLIM5 protein were able to protect F-actin from depolymerization in vitro in the presence of Lat B (an F-actin depolymerizer). Our results contribute to a better understanding of the biochemical role of GhWLIM5 in modulating the dynamic F-actin network in cotton.

Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes

Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

Rhodopsin–EGFP knock-ins for imaging quantal gene alterations

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin–EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.

380. Studies on the Mechanism of Gene Repair Using Saccharomyces cerevisae as a Model System

Oligonucleotides are being used to direct base changes within genes and can be used as a gene therapy for human disease. While the entire mechanism of the targeted nucleotide exchange (TNE) reaction has yet to be elucidated, there is evidence that it may be separated into at least two phases: the pairing phase, which consists of the MSSO pairing to the target site within the gene, and the repair phase, in which the MSSO directs the repair of the targeted base. This repair phase could possibly be separated into two phases: the first repair event, which is the correction of the strand to which the oligonucleotide is complementary, and the second repair event, in which the corrected strand serves as a template to repair the non-targeted strand. While the proteins that are involved in the pairing phase of the reaction are known, it remains unclear as to which proteins are playing a role in the repair phase of the reaction. Yeast strains deficient in Rad1p and MMR proteins, Msh2p and Msh6p, were used to determine the ability of a modified single-stranded oligonucleotide to direct a base change within a mutant Hygromycin eGFP fusion gene. The data indicate that in the absence of these proteins, the oligonucleotide is incapable of directing the base change, as the targeting frequency is reduced 30 fold and shows very little targeting activity. Upon complementation of Rad1p, using the 2um based plasmid pNF1000, the ability to direct the base change is restored. These data indicate that proteins involved in MMR and NER are playing a role in the TNE reaction and future studies aim to elucidate this role.