Cmv and sv40

The host-specific infection of Avian Astrovirus (AAstVs) has posed significant challenges to the poultry industry, resulting in substantial economic losses. However, few reports exist on the functional consequences of genome diversity, cross-species infectivity and mechanisms governing virus replication of AAstVs, making it difficult to develop measures to control astrovirus transmission. Reverse genetics technique can be used to study the function of viruses at the molecular level, as well as investigating pathogenic mechanisms and guide vaccine development and disease treatment...

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs...

The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot...

Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide, accounting for 24.5% of total new cancer cases and 15.5% of total cancer deaths. Similarly, BC is the most common cancer among Moroccan women, comprising a noteworthy percentage of 40% of all cancers in women. Globally, 15% of cancers are attributable to infections; among them, viruses play a significant role. The present study aimed to explore the presence of a wide range of viral DNA in samples recovered from 76 Moroccan patients with BC and 12 controls using Luminex technology...

The establishment of cell lines with a high protein production is the most crucial objective in the field of biopharmaceuticals. To this end, efforts have been made to increase transgene expression through promoter improvement, but the efficiency or stability of protein production was insufficient for use in commercial production. Here, we developed a novel strategy to increase the efficiency and stability of protein production by hybridizing a promoter that exhibits higher expression levels at the transient level with a promoter that exhibits higher stability at the stable level...

Ongoing research efforts to identify potent regulatory sequences that deliver robust and sustained transgene expression are critical for Chinese hamster ovary (CHO) cell line development technologies to meet the growing demand for recombinant proteins. Here we report the engineering and validation of a highly customizable single vector toolkit that comprises an all-in-one dual luciferase reporter system for quantitative and systematic interrogation of transcriptional regulatory sequences in transient and stable transfectants of CHO cells...

Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1α, SV40, and TK promoters, into the well-known genomic safe harbor site, AAVS1. EGFP expression driven by the CMV promoter increased up to 1.5-fold at 32 °C versus 37 °C under stable expression, while others showed no hypothermic response...

Herein, a Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation level in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail...

BACKGROUND: Zika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP. METHODS: A PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP...

Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP , CXCL12 , IGFBP2 , CTGF , JAG1 , SNAI1 , and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting...

It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression...

Karyomegalic interstitial nephritis (KIN) is a rare genetic kidney disease associated with a mutation in FAN1 gene and is often underdiagnosed. The histomorphology demonstrates chronic interstitial nephritis with tubular epithelial cells showing bizarre enlarged nuclei. We present a case report of a 47-year-old multiparous South-Indian woman presenting with bilateral pitting pedal oedema and mild hypertension. At the time of presentation, her serum creatinine was 1.52 mg/dL and urine analysis showed mild proteinuria...

BACKGROUND: Glioma is the most common primary brain tumor, occurring due to the carcinogenesis of glial cells in the brain and spinal cord. Many aspects of the mechanism of its tumorigenesis remain unknown. The relationship between viral infection and glioma is one of the most important research aspects in this field. Currently, there is a lack of systematic reviews and meta-analyses to evaluate the effect of viral infection on the prognosis of glioma patients. The purpose of this study was to evaluate the relationship between viral infection and the prognosis of glioma patients, aimed at evaluating the prognostic value of the detection of viral infection...

Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human genomic safe harbors (GSHs)-AAVS1, CCR5, and human ROSA26 loci-were evaluated with respect to the transgene expression level and stability in human embryonic kidney (HEK293) cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity...

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty ( SB100X ) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV- SB100X -SV40 plasmids...

Obstructive Sleep Apnea is characterized by interrupted breathing that leads to cardiovascular sequelae including chronic hypertension that can persist into waking hours. Chronic intermittent hypoxia, which models the hypoxemia associated with sleep apnea, is sufficient to cause a sustained increase in blood pressure that involves the central nervous system. The median preoptic nucleus (MnPO) is an integrative forebrain region that contributes to blood pressure regulation and neurogenic hypertension. The MnPO projects to the paraventricular nucleus (PVN), a pre-autonomic region...

To establish a stable and scalable transient protein production system, we modified the EF-1 first intron size and verified the order of two recombinant enhancers downstream of the SV40 polyA sequence. This new vector was named pHH-Gemini (pHH-GM1) and was used to express alpha kinase 1 (ALPK1) and various other proteins, NLRP3, F-actin, Camodulin, PP2A, URAT1, Rab11a and myosin IIA. The results showed that, compared with six commercial plasmids, pHH-GM1 significantly enhanced His-HA-ALPK1 expression in a western blot analysis of transfected HEK293T cells...

OBJECTIVE: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells. METHODS: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry...

BACKGROUND Vectors are widely used to drive gene expression using a promoter. However, not all promoters are able to drive ectopic gene expression efficiently, including CMV promoter. Here, we report our data using CMV promoter for high-level gene expression in a B lymphoma cell line DG75. MATERIAL AND METHODS A plasmid (pcDNA3.1(+)) containing the CD21 gene driven under CMV promoter was constructed. The plasmid was stably transfected into a human B lymphoma cell line DG75 for cellular surface CD21 expression, and flow cytometry was used to monitor CD21 expression...

BACKGROUND: Chordomas are rare tumors assumed to derive from notochordal remnants. We believe that a molecular switch is responsible for their malignant behavior. The involvement of oncogenic viruses has not been studied, however. Thus, in the present study, we investigated the presence of oncogenic viruses in chordomas. METHODS: DNA and RNA from snap-frozen chordoma (n = 18) and chondrosarcoma (n = 15) specimens were isolated. Real-time PCR or RT-PCR was performed to assess the presence of multiple oncogenic viruses, including herpesviridea (herpes simplex virus [HSV]-1, HSV-2, Epstein-Barr virus [EBV], cytomegalovirus, human herpesvirus [HHV]- 6, HHV-7, and Kaposi's sarcoma-associated herpesvirus), polyomaviridea (parvovirus B19 [PVB19], BK virus, JC virus, Simian virus 40, Merkel cell polyomavirus, human polyomavirus [HPyV]-6, and HPyV-7), papillomaviridae, and respiratory viruses...