To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock-out and screening of drug resistant ES cell clones. To construct HygR-neoR expression vector, pTK-hygR-pA and PGK-neoR-pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn I and Xba I digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hygR and neoR gene transcripts were detected by RT-PCR. 7 founder mice carrying hyg-neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hygR and neoR gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti-drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.
Animals ; Cinnamates ; pharmacology ; Drug Resistance, Multiple ; genetics ; Fibroblasts ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mice ; Mice, Transgenic ; Neomycin ; pharmacology ; Transgenes ; genetics

To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Ganciclovir ; pharmacology ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Goats ; Integrases ; genetics ; Neomycin ; pharmacology ; Phosphotransferases ; genetics ; metabolism

Oxidative stress plays an important role in the pathogenesis of aminoglycoside-induced hearing loss and represents a promising target for treatment. We tested the potential effect of apigenin, a natural flavonoid with anticancer, anti-inflammatory, and antioxidant activities, on neomycin-induced ototoxicity in cochlear hair cells in vitro. Results showed that apigenin significantly ameliorated the loss of hair cells and the accumulation of reactive oxygen species upon neomycin injury. Further evidence suggested that the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway was activated by apigenin treatment. Disruption of the Nrf2 axis abolished the effects of apigenin on the alleviation of oxidative stress and subsequent apoptosis of hair cells. This study provided evidence of the protective effect of apigenin on cochlear hair cells and its underlying mechanism.
Apigenin/pharmacology* ; Apoptosis ; Hair Cells, Auditory/metabolism* ; Humans ; NF-E2-Related Factor 2/pharmacology* ; Neomycin ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Signal Transduction

Mastoparan is an amphiphilic tetradecapeptide derived from wasp venom which activates G-proteins. Several secondary effects have been attributed to this peptide, including activation of phospholipase and phosphatidylinositol kinase. The aim of the present study was to investigate the effects of mastoparan on vascular contractility. Rabbit aortic rings were cut and mounted on a force transducer to record isometric tension on a polygraph. The effects of mastoparan were then investigated on the contractile responses in the isolated rabbit aorta with or without endothelium. The results were summarized as follows; 1. Mastoparan caused biphasic response, a transient relaxation followed by a further contraction, in norepinephrine (NE)-precontracted ring with endothelium. These effects were not observed in the aorta in the absence of endothelium. 2. Mastoparan-induced transient relaxation was significantly inhibited by treatment with a N-omega-nitro-L-arginine or methylene blue. 3. When an inhibitor of phospholipase C, neomycin was added to the precontracted aortic ring with NE, the transient relaxation induced by mastoparan was inhibited, but sustained contraction was not inhibited. 4. When an inhibitor of phospholipase A2, quinacrine and inhibitor of the cyclooxygenase pathway, indomethacin, were added to a precontracted ring with NE, the transient relaxation induced by mastoparan was not inhibited, but sustained contraction was inhibited. 5. Mastoparan induced a contraction of the aorta either with or without endothelium. Indomethacin and nifedipine inhibited mastoparan-induced contraction. From the above results, we concluded that mastoparan acts on the endothelium and modifies the release of endothelium-derived relaxing factors such as nitric oxide and also endothelium-derived contracting factors such as metabolites of arachidonic acid.
Animal ; Aorta/*drug effects/physiology ; Arginine/analogs & derivatives/pharmacology ; Calcium/metabolism ; In Vitro ; Indomethacin/pharmacology ; Neomycin/pharmacology ; Nitroarginine ; Quinacrine/pharmacology ; Rabbits ; Support, Non-U.S. Gov't ; Vasoconstriction/*drug effects ; Wasp Venoms/*pharmacology

Neomycin is one of the aminoglycoside antibiotics, and on the cellular level it inhibits phospholipase C. The effects of neomycin on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma (PC12) cells by using the whole-cell clamp technique. The I(ACh) on PC12 cells proved to be generated through activation of the neuronal nicotinic receptor. ACh (30 micromol/L) induced an inward current at a holding potential of -80 mV. When the cells were applied with neomycin (0.01~1 mmol/L) and ACh (30 micromol/L) simultaneously, an inhibitory effect of neomycin on the peak of I(ACh) was observed. This effect was fast, reversible and concentration-dependent. Pretreatment with neomycin for 3~8 min had no influence on its inhibitory effect. Activation of protein kinase C by using an exogenous activator exerted an inhibitory action on I(ACh). However, intracellular dialysis with a PKC inhibitor (PKCI 19-31, 0.1~5 micromol/L) did not affect the inhibitory effect of neomycin. The results obtained suggest that neomycin exerts an inhibitory effect on I(ACh) without involvement of the blockage of phospholipase C.
Animals ; Membrane Potentials ; drug effects ; Neomycin ; pharmacology ; PC12 Cells ; Patch-Clamp Techniques ; Protein Kinase C ; metabolism ; Rats ; Receptors, Nicotinic ; drug effects ; metabolism ; physiology

Homologous recombination is an important technique that is used to modify mammalian genome. Here, we constructed an efficient common gene targeting vector based on the plasmid pBS246. The vector consisted two positive selection markers, neomycin resistance gene (neo) and enhanced green fluorescent protein gene (EGFP) flanked by locus of X-over P1 (LoxP) sites. Two synthesized multiple cloning sequences MCS-1 and MCS-2 that contain several "8 bp cutter" enzyme sites were placed in outside of LoxP sites. Additionally, a negative selection marker HSV-tk (herpes simplex virus thymidine kinase) gene was located adjacent to MCS-1 site. The constructed vector was named pGT-V1, and its functions were characterized in C2C12 cells. The vector had the following unique features: 1) EGFP was used to monitor instantly the transfection rate that was essential for increasing the efficiency of gene knockout (KO); 2) The EGFP marker located between two LoxP sites was able to be removed from KO positive cells to avoid the potential damage of selection markers to the recipient cells. The process could be monitored visually and the positive cells without selecting markers (the loss of green fluorescent cells) could be sorted out by either flow cytometry or immunomagnetic beads; 3) "8 bp cutter" restriction sites were embedded in MCS sequences, which then enhanced the versatility of this vector. In summary, the constructed plasmid optimized the vector of gene targeting and provided a new technique means for the transgenic animal research.
Animals ; Base Sequence ; Cloning, Molecular ; Drug Resistance ; genetics ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Homologous Recombination ; Mice ; Molecular Sequence Data ; Neomycin ; pharmacology

PURPOSE: microRNAs (miRNAs) are non-coding RNAs composed of 20 to 22 nucleotides that regulate development and differentiation in various organs by silencing specific RNAs and regulating gene expression. In the present study, we show that the microRNA (miR)-183 cluster is upregulated during hair cell regeneration and that its inhibition reduces hair cell regeneration following neomycin-induced ototoxicity in zebrafish. MATERIALS AND METHODS: miRNA expression patterns after neomycin exposure were analyzed using microarray chips. Quantitative polymerase chain reaction was performed to validate miR-183 cluster expression patterns following neomycin exposure (500 µM for 2 h). After injection of an antisense morpholino (MO) to miR-183 (MO-183) immediately after fertilization, hair cell regeneration after neomycin exposure in neuromast cells was evaluated by fluorescent staining (YO-PRO1). The MO-183 effect also was assessed in transgenic zebrafish larvae expressing green fluorescent protein (GFP) in inner ear hair cells. RESULTS: Microarray analysis clearly showed that the miR-183 cluster (miR-96, miR-182, and miR-183) was upregulated after neomycin treatment. We also confirmed upregulated expression of the miR-183 cluster during hair cell regeneration after neomycin-induced ototoxicity. miR-183 inhibition using MO-183 reduced hair cell regeneration in both wild-type and GFP transgenic zebrafish larvae. CONCLUSION: Our work demonstrates that the miR-183 cluster is essential for the regeneration of hair cells following ototoxic injury in zebrafish larvae. Therefore, regulation of the miR-183 cluster can be a novel target for stimulation of hair cell regeneration.
Animals ; Animals, Genetically Modified ; Cell Count ; Gene Expression Profiling ; Gene Expression Regulation/drug effects ; Gene Knockdown Techniques ; Green Fluorescent Proteins/metabolism ; Hair Cells, Auditory/drug effects ; Hair Cells, Auditory/physiology ; Larva/drug effects ; Larva/genetics ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Morpholinos/pharmacology ; Neomycin/toxicity ; Regeneration/drug effects ; Regeneration/genetics ; Zebrafish/genetics