High Capacity Vectors

Bajpai, Bhakti

doi:10.1007/978-81-322-1554-7_1

Cited by 6 publications

(5 citation statements)

References 3 publications

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“…BACs have been used to some degree of success with mice while studying neurological diseases such as PD [63]. BACs are preferred for these kinds of genetic studies because they accommodate much larger sequences without the risk of rearrangement and are therefore more stable than other types of cloning vectors [5]. However, the enhancement of expression may result in the random integration of multiple copies of the transgene [63].…”

Section: Discussionmentioning

confidence: 99%

Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

Chen

2022

J Biomed Sci

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Background Parkinson’s disease (PD) is one of the most important neurodegenerative disorders in elderly people. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are found in a large proportion of the patients with sporadic and familial PD. Mutations can occur at different locations in the LRRK2. Patients with LRRK2 ROC-COR mutations face an increased risk of typical motor symptoms of PD, along with cognitive decline. An animal model with a monogenic LRRK2 gene mutation is a suitable model for exploring the pathophysiology of PD and identifying potential drug therapies. However, the effect of homozygous (HOM) LRRK2 in PD pathophysiology is unclear. Methods We established human LRRK2 (hLRRK2) R1441G HOM transgenic (Tg) mice to explore the phenotype and pathological features that are associated with hLRRK2 R1441G Tg mouse models and discuss the potential clinical relevance. The open field test (OFT) was performed to examine motor and nonmotor behaviors. A CatWalk analysis system was used to study gait function. [18F]FDOPA PET was used to investigate functional changes in the nigrostriatal pathway in vivo. Transmission electron microscopy was used to examine the morphological changes in mitochondria and lysosomes in the substantia nigra. Results The R1441G HOM Tg mice demonstrated gait disturbance and exhibited less anxiety-related behavior and exploratory behavior than mice with hLRRK2 at 12 months old. Additionally, [18F]FDOPA PET showed a reduction in FDOPA uptake in the striatum of the HOM Tg mice. Notably, there was significant lysosome and autophagosome accumulation in the cytoplasm of dopaminergic neurons in R1441G hemizygous (HEM) and HOM mice. Moreover, it was observed using transmission electron microscopy (TEM) that the mitochondria of R1441G Tg mice were smaller than those of hLRRK2 mice. Conclusion This animal provides a novel HOM hLRRK2 R1441G Tg mouse model that reproduces some phenotype of Parkinsonism in terms of both motor and behavioral dysfunction. There is an increased level of mitochondrial fission and no change in the fusion process in the group of HOM hLRRK2 R1441G Tg mouse. This mutant animal model of PD might be used to study the mechanisms of mitochondrial dysfunction and explore potential new drug targets.

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Section: Discussionmentioning

confidence: 99%

Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

Chen

2022

J Biomed Sci

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“…Although the use of RMCE is promising, particularly for inserting large transgenes, the method has a few limitations: First, insertions are generated at relatively low frequency (one in three injected animals). Second, co-insertion of the large self-excising cassette (7.7 kb) limits the size of transgene that can be inserted since most general cloning plasmids are limited to carrying 15 kb inserts (Bajpai 2014). Third, attempts by Nonet (2020) to integrate arrays were unsuccessful, possibly because constitutive expression of Flp recombinase "destroyed" arrays.…”

Section: Mosti In Comparison To Commonly Used Insertion Methodsmentioning

confidence: 99%

Modular safe-harbor transgene insertion (MosTI) for targeted single-copy and extrachromosomal array integration in C. elegans

Mouridi

Alkhaldi

Frøkjær-Jensen

2022

Preprint

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Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here we describe a modular safe harbor transgene insertion (MosTI) for use in C. elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by non-homologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25 - 75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7 - 44%) at MosTI landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for eight integrated arrays (6 to 37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of one array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

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“…BACs are artificial DNA constructs dependent on a functional F-plasmid employed for cloning huge DNA in Escherichia coli (E. coli) [77,78]. F-plasmids are used to synthesize BACs because they permit equal and even distribution of the plasmids and their viral inserts in multiplying bacteria.…”

Section: Bacterial Artificial Chromosomes (Bacs)mentioning

confidence: 99%

The Quest for Immunity: Exploring Human Herpesviruses as Vaccine Vectors

Kamel,

Munds,

Verma

2023

IJMS

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Herpesviruses are large DNA viruses that have long been used as powerful gene therapy tools. In recent years, the ability of herpesviruses to stimulate both innate and adaptive immune responses has led to their transition to various applications as vaccine vectors. This vaccinology branch is growing at an unprecedented and accelerated rate. To date, human herpesvirus-based vectors have been used in vaccines to combat a variety of infectious agents, including the Ebola virus, foot and mouth disease virus, and human immunodeficiency viruses. Additionally, these vectors are being tested as potential vaccines for cancer-associated antigens. Thanks to advances in recombinant DNA technology, immunology, and genomics, numerous steps in vaccine development have been greatly improved. A better understanding of herpesvirus biology and the interactions between these viruses and the host cells will undoubtedly foster the use of herpesvirus-based vaccine vectors in clinical settings. To overcome the existing drawbacks of these vectors, ongoing research is needed to further advance our knowledge of herpesvirus biology and to develop safer and more effective vaccine vectors. Advanced molecular virology and cell biology techniques must be used to better understand the mechanisms by which herpesviruses manipulate host cells and how viral gene expression is regulated during infection. In this review, we cover the underlying molecular structure of herpesviruses and the strategies used to engineer their genomes to optimize capacity and efficacy as vaccine vectors. Also, we assess the available data on the successful application of herpesvirus-based vaccines for combating diseases such as viral infections and the potential drawbacks and alternative approaches to surmount them.

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High Capacity Vectors

Cited by 6 publications

References 3 publications

Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

Modular safe-harbor transgene insertion (MosTI) for targeted single-copy and extrachromosomal array integration in C. elegans

The Quest for Immunity: Exploring Human Herpesviruses as Vaccine Vectors

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