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Expression vector

Erqing Jin, Lynn Wong, Yun Jiao, Jake Engel, Benjamin Holdridge, Peng Xu
Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering. Along the way, combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits. In this report, we present an efficient evolutionary approach to build a range of regulatory control elements...
December 2017: Synthetic and Systems Biotechnology
Harrison C Brown, Philip M Zakas, Stephan N George, Ernest T Parker, H Trent Spencer, Christopher B Doering
Potency is a key optimization parameter for hemophilia A gene therapy product candidates. Optimization strategies include promoter engineering to increase transcription, codon optimization of mRNA to improve translation, and amino-acid substitution to promote secretion. Herein, we describe both rational and empirical design approaches to the development of a minimally sized, highly potent AAV-fVIII vector that incorporates three unique elements: a liver-directed 146-nt transcription regulatory module, a target-cell-specific codon optimization algorithm, and a high-expression bioengineered fVIII variant...
June 15, 2018: Molecular Therapy. Methods & Clinical Development
Legang Sun, Ling Liu, Tingting Yu, Qiuqin Wang, Honghai Fu
In the present study, RNA interference (RNAi) was used to investigate the effect of vascular cell adhesion molecule 1 ( VCAM1 ) silencing on the proliferation of human oral squamous carcinoma HN12 cells. HN12 cells were divided into three groups: The untreated blank control cell group (CK), the negative control group transfected with non-homologous vector (NC) and the positive group transfected with the target sequence VCAM1 small hairpin RNA (KD). Reverse-transcription polymerase chain reaction and western blot analysis were used to examine the effects of VCAM1 -knockdown on the mRNA expression of VCAM1 and subsequent protein expression...
April 2018: Oncology Letters
Bo Hu, Yuanyuan Ma, Yue Yang, Lijian Zhang, Haibo Han, Jinfeng Chen
Cluster of differentiation 44 (CD44), a marker for cancer stem cells, has been reported to be associated with poor prognosis in non-small cell lung cancer (NSCLC), but its involvement in tumor growth has not fully been elucidated. The present study explored the associations between CD44 expression and clinicopathological features using immunohistochemistry in 94 NSCLC cases, as well as proliferation in cells with aberrant expression of this protein. Overexpression of CD44 was achieved by transfecting H1299 cells with CD44 expression vectors, and inhibition of CD44 expression was performed by transfecting small interfering RNAs into A549 cells...
April 2018: Oncology Letters
Ding Shi, Yongpan Liu, Dong Wu, Xujun Hu
Helicobacter pylori , the primary causative agent of stomach cancer, is known to affect gastric mucin expression. However, the underlying molecular mechanisms mediating this H. pylori -dependent effect remain unknown. In the present study, the effect of exogenous expression of the H. pylori virulence factor, CagA, on mucin 5AC oligomeric muscus/gel-forming (MUC5AC) expression was investigated using an in vitro model of the gastric mucosa. AGS cells were either untreated or transfected by a vector control (pCDNA3...
April 2018: Oncology Letters
Liang Wei, Qi Wang, Yanfei Zhang, Cheng Yang, Hongxin Guan, Jianxin Jiang, Zhiyang Sun
Intracranial aneurysm (IA) is a localized dilation of the blood vessel. The present study was designed to explore the mechanisms of rupture of IA. GSE13353 (including 11 ruptured and 8 unruptured IA samples) and GSE15629 (including 8 ruptured and 6 unruptured IA samples) were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) identified using limma and MetaDE packages were merged, and a protein-protein interaction (PPI) network analysis was performed using Cytoscape software...
April 2018: Oncology Letters
Jingxin Zhang, Guodong Deng, Lili Qiao, Hui Luo, Qiqi Liu, Ning Liang, Jian Xie, Jiandong Zhang
Galectin-3 is a multifunctional β-galactoside binding lectin associated with tumor progression. Previous studies confirmed the roles of galecin-3 overexpression and silencing in the biological behavior of Eca109 human esophageal cancer (EC) cells; galectin-3 may serve a critical role in the vasculogenic mimicry (VM) of tumors. Therefore, the present study examined the effects of galectin-3 knockdown using lentivirus vectors on VM in EC. Eca109 and EC9706 EC cells were transfected with a lentiviral vector to inhibit galectin-3 expression, or a control vector...
April 2018: Oncology Letters
Shuai Zhang, Yong Cao, Hao Cheng
Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification...
December 2017: Saudi Journal of Biological Sciences
Hongfang Guo, Rajwali Khan, Sayed Haidar Abbas Raza, Yue Ning, Dawei Wei, Sen Wu, Seyed Mahdi Hosseini, Irfan Ullah, Matthew D Garcia, Linsen Zan
The Krüppel-like factors (KLF) family plays an important role in adipogenesis, which is subject to internal hierarchical regulation. KLF3 is a member of KLF family, mainly responsible for adipocyte differentiation and fat deposition. However, the transcriptional regulation of bovine KLF3 gene and its relationship with KLF15 gene remains unclear during bovine adipogenesis. Here, we report that the expression pattern of KLF3 and KLF15 genes during bovine adipogenesis, when KLF15 gene was overexpressed through adenoviral vector (Ad-KLF15) in bovine adipocytes the expression level of KLF3 gene was increased, similarly when KLF15 was down regulated through siRNA the expression level of KLF3 was also reduced...
March 15, 2018: Gene
Peter Christ, Sharon R Hill, Joachim Schachtner, Frank Hauser, Rickard Ignell
Mosquito blood feeding transiently inhibits sugar- and host seeking through neuropeptide signaling. Short neuropeptide F (sNPF) is one of the neuromodulators involved in this regulation. Here, we identified the genes for the sNPF precursor and the sNPF receptor in the southern house mosquito, Culex quinquefasciatus. Comparative analyses are made with the genes of the sNPF precursor and receptor from two other important vectors, Aedes aegypti and Anopheles coluzzii. We functionally characterized the receptors in all three species using endogenous neuropeptides, and quantified their transcript expression following a blood meal and a sugar meal...
March 14, 2018: Peptides
Haimanti Mandal, Sameer S Katiyar, Rajan Swami, Varun Kushwah, Parmeshwar B Katare, Anand Kumar Meka, Sanjay K Banerjee, Amirali Popat, Sanyog Jain
The present work addresses the development and characterization of ε-Poly-l-Lysine/pDNA polyplexes and evaluation for their improved transfection efficacy and safety as compared to polyplexes prepared using Poly-l-Lysine and SuperFect®. Self-assembling polyplexes were prepared by varying the N/P ratio to obtain the optimum size, a net positive zeta potential and gel retardation. The stability in presence of DNase I and serum was assured using gel retardation assay. Their appreciable uptake in MCF-7 and 3...
March 14, 2018: International Journal of Pharmaceutics
Astrid Kritzinger, Boris Ferger, Frank Gillardon, Birgit Stierstorfer, Gerald Birk, Stefan Kochanek, Thomas Ciossek
Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice...
February 16, 2018: Neurobiology of Aging
Jie Yu, Yaoming Li, Maohua Zhong, Jingyi Yang, Dihan Zhou, Bali Zhao, Yuan Cao, Hu Yan, Ejuan Zhang, Yi Yang, Zhengshan Feng, Xiuwen Qi, Huimin Yan
The development of an effective HIV-1 vaccine is still a global priority. In recent years, vaccinia virus (VV) has been widely used as an HIV-1 vaccine vector, but its immune efficacy against HIV-1 antigens needs to be optimized. The extracellular enveloped virus (EEV) of VV is capable of faster entry, earlier release, and long-range dissemination. We hypothesized that an improvement in EEV formation by the manipulation of VV genes involved in the EEV release would consequently cause an improved expression of the VV carrying HIV-1 Env antigen and a subsequent enhanced immune response...
March 14, 2018: Antiviral Research
Sarinthip Preedagasamzin, Tiwaporn Nualkaew, Tanjitti Pongrujikorn, Natini Jinawath, Ryszard Kole, Suthat Fucharoen, Natee Jearawiriyapaisarn, Saovaros Svasti
Repair of a splicing defect of β-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with β-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of βE -globin pre-mRNA and achieves a long-term therapeutic effect...
March 14, 2018: Biochemical and Biophysical Research Communications
Yongjie Liu, Fujun Hou, Zhaoying Qian, Xiaolin Liu
The clade B serpins contain predominantly intracellular proteins and were reported to be involved in multiple biological functions, especially in inflammation and immune system function. However, studies about the role of the invertebrate intracellular serpins in immune responses were still deficient. Therefore, this paper focused on the functional characterization of LvserpinB3 in white shrimp Litopenaeus vannamei. The pAc5.1-LvserpinB3-EGFP vector was transfected into Drosophila Schneider 2 (S2) cells to analyze the subcellular localization of LvserpinB3, and fluorescent imaging showed that LvserpinB3 were mainly localized to the mitochondria...
March 14, 2018: Developmental and Comparative Immunology
Yuhan Wang, Craig Dorrell, Willscott E Naugler, Michael Heskett, Paul Spellman, Bin Li, Feorillo Galivo, Annelise Haft, Leslie Wakefield, Markus Grompe
Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic β cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to β-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic β cells and whether different cell types have the same reprogramming potential...
February 21, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Charu Aggarwal, Andrew R Haas, Susan Metzger, Laura K Aguilar, Estuardo Aguilar-Cordova, Andrea G Manzanera, Gregoria Gómez-Hernández, Sharyn I Katz, Evan W Alley, Tracey L Evans, Joshua M Bauml, Roger B Cohen, Corey J Langer, Steven M Albelda, Daniel H Sterman
Gene-mediated cytotoxic immunotherapy (GMCI) is an immune strategy implemented through local delivery of an adenovirus-based vector expressing the thymidine kinase gene (aglatimagene besadenovec, AdV-tk) followed by anti-herpetic prodrug valacyclovir. A phase I dose escalation trial of GMCI followed by chemotherapy was conducted in patients with malignant pleural effusion (MPE). AdV-tk was administered intrapleurally (IP) in three cohorts at a dose of 1 × 1012 to 1013 vector particles. Primary endpoint was safety; secondary endpoints included response rate, progression-free survival, and overall survival...
February 21, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Yu Chen, Zhen Li, Zhu Xu, Huanyin Tang, Wenxuan Guo, Xiaoxiang Sun, Wenjun Zhang, Jian Zhang, Xiaoping Wan, Ying Jiang, Zhiyong Mao
The homologous recombination (HR) pathway is a promising target for cancer therapy as it is frequently upregulated in tumors. One such strategy is to target tumors with cancer-specific, hyperactive promoters of HR genes including RAD51 and RAD51C. However, the promoter size and the delivery method have limited its potential clinical applications. Here we identified the ~2.1 kb promoter of XRCC2, similar to ~6.5 kb RAD51 promoter, as also hyperactivated in cancer cells. We found that XRCC2 expression is upregulated in nearly all types of cancers, to a degree comparable to RAD51 while much higher than RAD51C...
March 16, 2018: Cell Death & Disease
Babak Barati, Firouz Ebrahimi, Shahram Nazarian
BACKGROUND: Cholera toxin (CT), responsible for the harmful effects of cholera infection, is made up of one A subunit (enzymatic), and five B subunits (cell binding). The release of cholera toxin is the main reason for the debilitating loss of intestinal fluid. Inhibition of the B subunit (CTB) may block CT activity. OBJECTIVE: To determine the effect of anti CTB-IgY against oral challenge with V. cholera in suckling infant mice. METHODS: The binding domain of cholera toxin was amplified and ligated into pET28a vector...
March 2018: Iranian Journal of Immunology: IJI
Elhussiny A Aboulnaga, Huibin Zou, Thorsten Selmer, Mo Xian
Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism...
March 13, 2018: Journal of Biotechnology
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