scholarly journals Efficiency of cell-type specific and generic promoters in transducing oxytocin neurons and monitoring their neural activity during lactation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Keerthi Thirtamara Rajamani ◽  
Amanda B. Leithead ◽  
Michelle Kim ◽  
Marie Barbier ◽  
Michael Peruggia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Neural Activity ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Viral Vector ◽  
Female Rats ◽  
Cell Populations ◽  
Specific Cell ◽  
Functional Studies ◽  
Drive Gene

AbstractHypothalamic oxytocin (OXT) and arginine-vasopressin (AVP) neurons have been at the center of several physiological and behavioral studies. Advances in viral vector biology and the development of transgenic rodent models have allowed for targeted gene expression to study the functions of specific cell populations and brain circuits. In this study, we compared the efficiency of various adeno-associated viral vectors in these cell populations and demonstrated that none of the widely used promoters were, on their own, effective at driving expression of a down-stream fluorescent protein in OXT or AVP neurons. As anticipated, the OXT promoter could efficiently drive gene expression in OXT neurons and this efficiency is solely attributed to the promoter and not the viral serotype. We also report that a dual virus approach using an OXT promoter driven Cre recombinase significantly improved the efficiency of viral transduction in OXT neurons. Finally, we demonstrate the utility of the OXT promoter for conducting functional studies on OXT neurons by using an OXT specific viral system to record neural activity of OXT neurons in lactating female rats across time. We conclude that extreme caution is needed when employing non-neuron-specific viral approaches/promoters to study neural populations within the paraventricular nucleus of the hypothalamus.

scholarly journals VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

2021 ◽  
Author(s):  
Arjun Khakhar ◽  
Cecily Wang ◽  
Ryan Swanson ◽  
Sydney Stokke ◽  
Furva Rizvi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tobacco Rattle Virus ◽  
Plant Size ◽  
Great Promise ◽  
Attractive Alternative ◽  
Gibberellin Biosynthesis ◽  
In Planta

Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work we developed a technology called VipariNama (ViN) in which vectors based on the Tobacco Rattle Virus (TRV) are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

scholarly journals Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ

Biology Open ◽  
2021 ◽  
Author(s):  
E.S. Zubkova ◽  
I.B. Beloglazova ◽  
E.I. Ratner ◽  
D.T. Dyikanov ◽  
K.V. Dergilev ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Cellular Response ◽  
Viral Vectors ◽  
Viral Vector ◽  
Ex Vivo ◽  
Human Adipose Tissue ◽  
Cell Types ◽  
Specific Cell ◽  
Therapeutic Benefits ◽  
Rat Adipose Tissue

Ex vivo, gene therapy is a powerful approach holding great promises for the treatment of both genetic and acquired diseases. Adeno-associated virus (AAV) vectors are safe and efficient delivery system for modification of mesenchymal stem cells (MSC) that could maximize their therapeutic benefits. Assessment to MSC viability and functional activity after infection with new AAV serotypes is necessary, due to AAV tropism to specific cell types. We infected human and rat adipose-tissue MSC with hybrid AAV-DJ serotype vectors carrying GFP and SCF genes. GFP expression from AAV-DJ was about 1.5-fold superior to that observed with AAV-2 and lasted for at least 21 days as was evaluated by flow cytometry and fluorescence microscopy. AAV-DJ proves to be suitable for the infection of rat and human MSC with a similar efficiency. Infected MSC were still viable however showing 25-30%. growth rate slowdown. Moreover, we found increase of SERPINB2 mRNA expression in human MSC whereas expression of other oxidative stress markers and extracellular matrix proteins was not affected. These results suggest that there is a differential cellular response in MSC infected with AAV viral vectors, which should be taken into account as it can affect the expected outcome for the therapeutic application.

Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 894-901 ◽  
Author(s):  
Christopher A. Klug ◽  
Samuel Cheshier ◽  
Irving L. Weissman
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter. Limiting dilution reconstitution of lethally irradiated recipient mice with 100% transduced, GFP+ LT-HSC showed that silencing of gene expression occurred rapidly in most integration events at the LT-HSC level, irrespective of the initial levels of GFP expression. When inactivation occurred at the LT-HSC level, there was no GFP expression in any hematopoietic lineage clonally derived from silenced LT-HSC. Inactivation downstream of LT-HSC that stably expressed GFPin long-term reconstituted animals was restricted primarily to lymphoid cells. These observations suggest at least 2 distinct mechanisms of silencing retrovirally expressed genes in hematopoietic cells.

Convergence of sensory pathways in the development of somatic and visceral hypersensitivity

2006 ◽  
Vol 291 (4) ◽  
pp. G658-G665 ◽  
Author(s):  
Klaus Bielefeldt ◽  
Kenneth Lamb ◽  
G. F. Gebhart
Keyword(s):  
Viral Vectors ◽  
Fluorescent Protein ◽  
Viral Vector ◽  
Thermal Hyperalgesia ◽  
Colorectal Distension ◽  
Sensory Pathways ◽  
Pain Syndromes ◽  
Transient Overexpression ◽  
Green Fluorescent ◽  
Thermal Hypersensitivity

Sensory neurons innervating different tissues converge onto second-order neurons in the spinal cord. We examined whether inflammation or transient overexpression of nerve growth factor (NGF) in one tissue triggers hypersensitivity in referral sites. Thresholds to mechanical and thermal stimulation of the hindpaw, visceromotor responses to colorectal distension, and cystometrograms were performed in appropriate controls and mice with experimentally induced cystitis, inflammation of the hindpaw or front paw, or injection of viral vectors encoding NGF or green fluorescent protein (GFP). Cystitis and NGF but not GFP overexpression in the bladder triggered bladder hyperactivity associated with mechanical and thermal hypersensitivity in cutaneous referral sites and enhanced responses to colorectal distension. Hindpaw inflammation and injection of the NGF- but not GFP-encoding viral vector or front paw inflammation induced mechanical and thermal hyperalgesia in the affected hindpaw and increased responses to colorectal distension without altering the micturition reflex. In conclusion, sensitization of sensory pathways by inflammation or NGF contributes to the development of hypersensitivity in neighboring organs and cutaneous referral sites and provides a potential mechanism underlying the coexistence of pain syndromes in patients with functional diseases.

scholarly journals Reprogramming of Human Huntington Fibroblasts Using mRNA

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Antje Arnold ◽  
Yahaira M. Naaldijk ◽  
Claire Fabian ◽  
Henry Wirth ◽  
Hans Binder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Vectors ◽  
Viral Vector ◽  
Ips Cells ◽  
Expression Array ◽  
Healthy Donors ◽  
Pluripotency Gene ◽  
Cell Sources ◽  
Induced Pluripotent

The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.

Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 894-901 ◽  
Author(s):  
Christopher A. Klug ◽  
Samuel Cheshier ◽  
Irving L. Weissman
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter. Limiting dilution reconstitution of lethally irradiated recipient mice with 100% transduced, GFP+ LT-HSC showed that silencing of gene expression occurred rapidly in most integration events at the LT-HSC level, irrespective of the initial levels of GFP expression. When inactivation occurred at the LT-HSC level, there was no GFP expression in any hematopoietic lineage clonally derived from silenced LT-HSC. Inactivation downstream of LT-HSC that stably expressed GFPin long-term reconstituted animals was restricted primarily to lymphoid cells. These observations suggest at least 2 distinct mechanisms of silencing retrovirally expressed genes in hematopoietic cells.

scholarly journals Peripheral blood lymphocytes as target cells of retroviral vector- mediated gene transfer

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1988-1997 ◽  
Author(s):  
F Mavilio ◽  
G Ferrari ◽  
S Rossini ◽  
N Nobili ◽  
C Bonini ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Gene Transfer ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Retroviral Vectors ◽  
Peripheral Blood Lymphocytes ◽  
Cell Populations ◽  
Target Cells ◽  
Specific Cell

Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T- cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

scholarly journals Viral vectors as a novel tool for clinical and neuropsychiatric research applications

2018 ◽  
Vol 31 (2) ◽  
pp. e000015 ◽  
Author(s):  
Yao Wang ◽  
Zhiwei Hu ◽  
Peijun Ju ◽  
Shan Yin ◽  
Fujie Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Vectors ◽  
Molecular Mechanisms ◽  
Gene Expression Regulation ◽  
Viral Vector ◽  
Neurological Diseases ◽  
Basic Research ◽  
Research Field ◽  
Research Fields ◽  
Pathogenic Genes

BackgroundA viral vector is a genetically modified vector produced by genetic engineering. As pathogenic genes in the virus are completely or largely eliminated, it is safe to be widely used in multidisciplinary research fields for expressing genes, such as neuroscience, metabolism, oncology and so on. Neuroscience and psychiatry are the most closely related disciplines in either basic research or clinical research, but the application of viral vectors in neuropsychiatry has not received much attention or not been widely accepted.ObjectiveThis article will focus on the application of viral vectors in basic and clinical neuropsychiatric research.MethodsBy using viral vectors, scientists can perform neurological labelling, gene expression regulation and physiological manipulation for investigating phenomenon from molecular mechanisms to behaviours. At the same time, to treat mental or neurological disorders, viral vectors can be designed for gene therapy, which alter gene expression levels or repair mutated genes in the brains of patients.PerspectiveViral vectors play an important role in basic research and clinical applications. To further understand brain function and prevent mental and neurological diseases, we hypothesize that viral vectors could be used along with various advanced technologies, such as sequencing and high-throughput expression analysis in the neuroscience research field.

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