scholarly journals Monosynaptic tracing success depends critically on helper virus concentrations

2019 ◽  
Author(s):  
Thomas K. Lavin ◽  
Lei Jin ◽  
Nicholas E. Lea ◽  
Ian R. Wickersham
Keyword(s):  
Viral Vectors ◽  
Deletion Mutant ◽  
First Generation ◽  
Viral Vector ◽  
Helper Virus ◽  
Neuronal Population ◽  
Wild Type ◽  
Practical Guidelines ◽  
Mouse Lines ◽  
Wild Type Control

ABSTRACTMonosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent “helper” adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.

scholarly journals Wild type AAV, recombinant AAV, and Adenovirus super infection impact on AAV vector mobilization

2020 ◽  
Author(s):  
Liujiang Song ◽  
R. Jude Samulski ◽  
Matthew L. Hirsch
Keyword(s):  
Particle Production ◽  
De Novo ◽  
Viral Vector ◽  
Production Systems ◽  
Helper Virus ◽  
Wild Type ◽  
Raav Production ◽  
Plasmid Transfection ◽  
Super Infection

AbstractRecombinant Adeno-associated viral vector (rAAV) mobilization is a largely theoretical process in which intact AAV vectors spread or “mobilize” from transduced cells and infect additional cells within, or external, of the initial host. This process can be replication independent (vector alone), or replication-dependent (de novo rAAV production facilitated by super-infection of both wild-type AAV (wtAAV) and Ad helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 Kb), rAAV-IDUA (3.7 Kb), and rAAV-NanoDysferlin (4.9 Kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and a rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be de fined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed non-replicative mobilization). In rAAV infected cells co-infected with Ad5 and wtAAV, rAAV particle production was increased > 50-fold compared to non-replicative conditions. In addition, replicative dependent rAAV vectors mobilized and resulted in >1,000 -fold transduction upon a subsequent 2nd round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization resistant vectors and the opportunity to derive better vector production systems.

scholarly journals “Self-inactivating” rabies viruses are susceptible to loss of their intended attenuating modification

2019 ◽  
Author(s):  
Makoto Matsuyama ◽  
Lei Jin ◽  
Thomas K. Lavin ◽  
Heather A. Sullivan ◽  
YuanYuan Hou ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Deletion Mutant ◽  
First Generation ◽  
Great Majority ◽  
Viral Particles ◽  
New Form ◽  
Transsynaptic Tracing ◽  
Circuit Function ◽  
Neuronal Physiology

SUMMARYAn article in Cell reported a new form of modified rabies virus that was apparently capable of labeling neurons “without adverse effects on neuronal physiology and circuit function” but that nevertheless was able to spread between neurons as efficiently as the widely-used first-generation deletion-mutant (ΔG) rabies viral vectors. The new “self-inactivating” rabies (“SiR”) viruses differed from first-generation vectors only by the addition of a destabilization domain to the viral nucleoprotein. We noticed that the transsynaptic tracing results from that article appeared inconsistent with the strategy described in it: specifically, the viruses were able to spread between neurons even in the absence of the exogenous protease that was meant to be required. We hypothesized that the viruses used were actually mutants that had lost the intended addition to the nucleoprotein, making them de facto first-generation viruses. We obtained samples of two SiR viruses from the authors and show here that the great majority of viral particles in both the “SiR-CRE” and “SiR-FLPo” samples were mutants that had lost the intended modification, consistent with our hypothesis. We also found that SiR-CRE killed 70% of infected neurons in vivo within two weeks, consistent with the prediction that mutants without the intended modification would share the toxic phenotype typical of first-generation rabies viral vectors. We hypothesize that the same or similar mutations were present in the viruses used in the original article and that this explains the paradoxical reported findings. While it may be possible to successfully make SiR viral preparations that are not dominated by such mutants, and while it may also be possible that such intact SiR viruses are indeed nontoxic to neurons, we predict that it will not be possible to replicate the transsynaptic tracing results from the original paper unless using mutants similar to the ones that we report here.

scholarly journals Presence of Arp Specifically Contributes to Joint Tissue Edema Associated with Early-Onset Lyme Arthritis

2013 ◽  
Vol 82 (1) ◽  
pp. 43-51 ◽  
Author(s):  
Petronella R. Hove ◽  
Gary J. Haldorson ◽  
Forgivemore Magunda ◽  
Troy Bankhead
Keyword(s):  
Early Onset ◽  
Deletion Mutant ◽  
Lyme Arthritis ◽  
Mutant Clone ◽  
Wild Type ◽  
Content Type ◽  
Immunodeficient Mice ◽  
Tissue Edema ◽  
Significant Difference ◽  
Wild Type Control

ABSTRACTAntiserum to theBorrelia burgdorferiarthritis-related protein, Arp, has been shown to prevent or reduce arthritis in immunodeficient mice. To directly investigate the requirement for this lipoprotein in the generation of Lyme arthritis, we utilized targeted deletion to generate aB. burgdorfericlone that lacked only thearpgene locus. Infection of Lyme disease-susceptible C3H/HeN mice with thearpdeletion mutant demonstrated significantly reduced tibiotarsal joint swelling during the first 6 weeks of infection compared to a wild-type control. The severity of joint swelling was restored to wild-type levels in mice infected with anarpmutant clone complemented incis. Interestingly, the reduced swelling of joint tissues exhibited by mice infected with thearpdeletion mutant did not directly correspond to reduced underlying arthritis. Histopathology data at 2 weeks postinfection showed some reduction in arthritis severity caused by thearpmutant clone; however, by 8 weeks, no significant difference was observed between joint tissues infected by the wild-type orarpmutant clones. The spirochete load in the joint tissues of mice infected with thearpmutant was found to be greater than that exhibited by the wild-type control. Our findings demonstrate that this lipoprotein contributes to the generation of early-onset joint swelling and suggests thatarpexpression has a negative secondary effect on total spirochete numbers in joint tissues.

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 Overexpression of Mucin 1 Suppresses the Therapeutical Efficacy of Disulfiram against Canine Mammary Tumor

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 37
Author(s):  
Ying Zhao ◽  
Zixiang Lin ◽  
Zhaoyan Lin ◽  
Chaoyu Zhou ◽  
Gang Liu ◽  
...  
Keyword(s):  
Mammary Tumor ◽  
Mammary Tumors ◽  
Expression Level ◽  
Canine Mammary Tumor ◽  
Wild Type ◽  
Mucin 1 ◽  
Mammary Tumor Cell ◽  
Mammary Tumor Cell Line ◽  
Canine Mammary Tumors ◽  
Wild Type Control

Mucin 1 (MUC1), a transmembrane protein, is closely associated with the malignancy and metastasis of canine mammary tumors; however, the role of overexpressed MUC1 in the development of cancer cells and response to drug treatment remains unclear. To address this question, we developed a new canine mammary tumor cell line, CIPp-MUC1, with an elevated expression level of MUC1. In vitro studies showed that CIPp-MUC1 cells are superior in proliferation and migration than wild-type control, which was associated with the upregulation of PI3K, p-Akt, mTOR, Bcl-2. In addition, overexpression of MUC1 in CIPp-MUC1 cells inhibited the suppressing activity of disulfiram on the growth and metastasis of tumor cells, as well as inhibiting the pro-apoptotic effect of disulfiram. In vivo studies, on the other side, showed more rapid tumor growth and stronger resistance to disulfiram treatment in CIPp-MUC1 xenograft mice than in wild-type control. In conclusion, our study demonstrated the importance of MUC1 in affecting the therapeutical efficiency of disulfiram against canine mammary tumors, indicating that the expression level of MUC1 should be considered for clinical use of disulfiram or other drugs targeting PI3K/Akt pathway.

scholarly journals Mating in Saccharomyces cerevisiae: The Role of the Pheromone Signal Transduction Pathway in the Chemotropic Response to Pheromone

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 19-32 ◽  
Author(s):  
Kathrin Schrick ◽  
Barbara Garvik ◽  
Leland H Hartwell
Keyword(s):  
Signal Transduction ◽  
Map Kinase ◽  
Transcriptional Activation ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Wild Type ◽  
Mating Partner ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Wild Type Control

Abstract The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the β or γ subunit (ste4 and stel8) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

scholarly journals How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

2021 ◽  
Vol 22 (14) ◽  
pp. 7545
Author(s):  
Myriam Sainz-Ramos ◽  
Idoia Gallego ◽  
Ilia Villate-Beitia ◽  
Jon Zarate ◽  
Iván Maldonado ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Clinical Practice ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Viral Vector ◽  
Clinical Success ◽  
Genetic Material ◽  
Viral Gene ◽  
Promising Alternative ◽  
Vector Systems

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

scholarly journals Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Madhavi Latha Gandla ◽  
Niklas Mähler ◽  
Sacha Escamez ◽  
Tomas Skotare ◽  
Ogonna Obudulu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Biomass Production ◽  
Enzymatic Saccharification ◽  
Dry Weight ◽  
Populus Tremula ◽  
Transgenic Trees ◽  
Wild Type ◽  
Glucose Yield ◽  
Transgenic Lines ◽  
Wild Type Control

Abstract Background Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth. Results In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20–44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26–50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control. Conclusions The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

scholarly journals α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 771
Author(s):  
Julen Rodríguez-Castejón ◽  
Ana Alarcia-Lacalle ◽  
Itziar Gómez-Aguado ◽  
Mónica Vicente-Pascual ◽  
María Ángeles Solinís Aspiazu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fabry Disease ◽  
Viral Vector ◽  
Viral Gene ◽  
Lysosomal Storage ◽  
Wild Type ◽  
Dose Administration ◽  
Viral Gene Therapy ◽  
Liver Transaminases ◽  
Low Levels

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

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