Cloning of the GH gene from the beluga sturgeon (Huso huso) into a lentivial & none viral constructs and it’s expression in HEK cell lines

Caviar-producing fish with their economically valuable product are important in fisheries. The cDNA growth hormone (GH) of Beluga sturgeon (Huso huso) was constructed using total RNA from pituitary glands. To construct the recombinant and active lentiviruses carring GH gene, this DNA sequence was inserted into the cloning vector pTZ57R/T and subsequently cutted from pTZ57R/T by endonuclease enzyme and incorporated into lentivirus vector pNL-EGFP/CMV-WPRE on upstream of an IRES cassette. We also insert a reporter EGFP gene downstream of IRES so transfection and transduction steps can be traced. Using this vector plus virus packaging and envelope vectors, HEK293T cells was co-transfected by DNA-Lipofectamine complexes method. Cell supernatant full of virions was collected 48 hours later and concentrated using Amicon columns to obtain a high-titer virus stock. Nearly 1/5 of this stock was applied to a new batch of cultured HEK-293T. After 72h expression of EGFP gene was detected and the cells was collected for further analysis. Total RNA of these transduced cells was extracted and GH mRNA expression was revealed by RT-PCR. Results showed that, lentiviral vectors (LV) as a gene transfer system provide efficient delivery, integration and long-term expression by establishing a stable provirus in target cells and could be important tool in aquaculture and fisheries biotechnology research to increase the growth rate of farmed fish by transferring growth hormone (GH) transgenes into fish

Cloning of the GH gene from the beluga sturgeon (Huso huso) into a lentivial & none viral constructs and its expression in HEK cell lines

Caviar-producing fish with their economically valuable product are important in fisheries. The cDNA growth hormone (GH) of Beluga sturgeon (Huso huso) was constructed using total RNA from pituitary glands. To construct the recombinant and active lentiviruses carring GH gene, this DNA sequence was inserted into the cloning vector pTZ57R/T and subsequently cutted from pTZ57R/T by endonuclease enzyme and incorporated into lentivirus vector pNL-EGFP/CMV-WPRE on upstream of an IRES cassette. We also insert a reporter EGFP gene downstream of IRES so transfection and transduction steps can be traced. Using this vector plus virus packaging and envelope vectors, HEK293T cells was co-transfected by DNA-Lipofectamine complexes method. Cell supernatant full of virions was collected 48 hours later and concentrated using Amicon columns to obtain a high-titer virus stock. Nearly 1/5 of this stock was applied to a new batch of cultured HEK-293T. After 72h expression of EGFP gene was detected and the cells was collected for further analysis. Total RNA of these transduced cells was extracted and GH mRNA expression was revealed by RT-PCR. Results showed that, lentiviral vectors (LV) as a gene transfer system provide efficient delivery, integration and long-term expression by establishing a stable provirus in target cells and could be important tool in aquaculture and fisheries biotechnology research to increase the growth rate of farmed fish by transferring growth hormone (GH) transgenes into fish

MRP1 gene expression level regulates the death and differentiation response of neuroblastoma cells

We have previously reported a strong correlation between poor prognosis in childhood neuroblastoma (NB) patients and high-level expression of the transmembrane efflux pump, Multidrug Resistance-associated Protein (MRP1), in NB tumour tissue. In this study, we inhibited the endogenous expression of MRP1 in 2 different NB tumour cell lines by stably transfecting an MRP1 antisense expression vector (MRP-AS). Compared with control cells, MRP-AS transfectant cells demonstrated a higher proportion of dead and morphologically apoptotic cells, spontaneous neuritogenesis, and, increased synaptophysin and neurofilament expression. Bcl-2 protein expression was markedly reduced in MRP-AS cells compared to controls. Conversely, we found that the same NB tumour cell line overexpressing the full-length MRP1 cDNA in sense orientation (MRP-S) demonstrated resistance to the neuritogenic effect of the differentiating agent, all-trans-retinoic acid. Taken together, the results suggest that the level of MRP1 expression in NB tumour cells may influence the capacity of NB cells for spontaneous regression in vivo through cell differentiation and death. © 2001 Cancer Research Campaign  http://www.bjcancer.co

An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice.

BACKGROUND & AIMS: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification. METHODS & RESULTS: Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time. CONCLUSION: These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases

Rat tyrosine hydroxylase promotor directs tetracycline-inducible foreign gene expression in dopaminergic cell types.: Mol.Brain Res.

NRC publication: Ye

Synergy Between Glutathione Peroxidase-1 and Astrocytic Growth Factors Suppresses Free Radical Generation and Protects Dopaminergic Neurons against 6-Hydroxydopamine

The degeneration of dopaminergic neurons in the course of Parkinson disease is largely blamed on oxidative damage in the brain. This study examined the potency of glutathione peroxidase-1 (GPX-1) to protect dopaminergic neurons against toxicity induced by the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). We generated pLV-GPX1, a recombinant lentivirus vector carrying the coding sequence for human GPX-1, into the SK-N-MC neuroblastoma cell line. The pLV-GPX1–infected neurons showed an over 3-fold increase in enzyme expression and a 2.6-fold increase in enzyme activity compared to the pLV-EGFP–infected control cells. In the pLV-GPX1–infected cells, we also detected significantly increased neuronal survival and resistance to 6-OHDA–mediated toxicity compared to our controls (75 ± 4% versus 51 ± 7%, p < 0.001). To maximize this protection, the neurons were treated with conditioned medium taken from growing primary astrocytes (astro-CM). We found the treated pLV-GPX1–infected neurons even more significantly resistant to 6-OHDA toxicity compared to their untreated counterparts (86 ± 5% versus 75 ± 4%, p < 0.001). Concomitant with increased neuroprotection, co-presence of overexpressed GPX-1 and astro-CM significantly increased glutathione (GSH) levels compared to when either of the two was present (p < 0.001). Further analysis showed nearly 2.7-fold reduction, in the presence of astro-CM, of hydrogen peroxide (H2O2) levels released from the pLV-GPX1–infected neurons compared to control groups (p < 0.001). Finally, regression analysis between H2O2 levels and cell viability showed that co-presence of GPX-1 and astro-CM reduced 33% of cell death rate (p < 0.05). These data highlight the antioxidant properties of GPX-1 in protecting dopaminergic neurons and further emphasize the capacity of astrocytes in pumping growth-inducing factors that may synergize with GPX-1 to accelerate neuroprotection

Crosstalk between breast cancer stem cells and metastatic niche: Emerging molecular metastasis pathway?

Metastatic colonization represents the final step of metastasis, and is the major cause of cancer mortality. Metastasis as an "inefficient" process requires the right population of tumor cells in a suitable microenvironment to form secondary tumors. Cancer stem cells are the only capable population of tumor cells to progress to overt metastasis. On the other hand, the occurrence of appropriate microenvironmental conditions within the target tissue would be critical for metastasis formation. Metastatic niche seems to be the specialized microenvironment to support tumor initiating cells at the distant organ. Master regulators not only determine cancer stem cell state, but also may have regulatory roles in metastatic niche elements. Meanwhile, both cancer stem cell and metastatic niche may function like two sides of the metastatic coin. Hypoxia inducible factors have multiple roles in regulation of both sides of this coin. TGF-β superfamily, also, have been considered as master regulators of epithelial to mesenchymal transition and metastasis and may play crucial roles in regulation of metastatic niche as well. In this regard, we hypothesize the presence of a possible emerging molecular pathway in the biological process of breast cancer metastasis. In this process, non-Smad TGF-β-induced metastasis connects cancer stem cell and metastatic niche formation through a central path, "Metastasis Pathway". © 2013 International Society of Oncology and BioMarkers (ISOBM)

SLUG and SOX9 Cooperatively Regulate Tumor Initiating Niche Factors in Breast Cancer

Presence of tumor initiating cells and a proper niche is essential for metastatic colonization. SLUG and SOX9 transcription factors play essential roles in induction and maintenance of tumor initiating capacity in breast cancer cells. On the other hand, Tenascin-C and Periostin are crucial factors in metastatic niche that support tumor initiating capability in breast cancer. In this study, regulatory effect of SLUG and SOX9 transcription factors on the expression of Tenascin-C and Periostin was examined. SLUG and SOX9 were overexpressed and knocked-down in MCF7 and MDA-MB-231 cells, respectively. The cells as little and highly invasive breast cancer-derived cells were infected by inducing and shRNA lentivirus constructs. Then, Tenascin-C and Periostin as well as SLUG and SOX9 expression levels were measured in the cells via Real-Time PCR. Simultaneous overexpression of SLUG and SOX9 significantly induced Tenascin-C and Periostin expression. SLUG and SOX9 knock-down also significantly reduced the expression of Tenascin-C and Periostin. In this analysis Periostin showed the most deviation in both up- and down-regulation levels. This regulatory effect might shed light to a crosstalk between factors involved in the tumor initiating capacity and metastatic niche of the breast cancer. © 2015, Springer Science+Business Media Dordrecht