scholarly journals Establishment of a Simple and Efficient Reverse Genetics System for Canine Adenoviruses Using Bacterial Artificial Chromosomes

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 767
Author(s):  
Hiromichi Matsugo ◽  
Tomoya Kobayashi-Kitamura ◽  
Haruhiko Kamiki ◽  
Hiroho Ishida ◽  
Akiko Takenaka-Uema ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Vectors ◽  
Reverse Genetics ◽  
Infectious Hepatitis ◽  
Recombinant Vaccines ◽  
Bacterial Artificial Chromosomes ◽  
Large Genome ◽  
Artificial Chromosomes ◽  
Genetic Modifications ◽  
Large Genome Size

Canine adenoviruses (CAdVs) are divided into pathotypes CAdV1 and CAdV2, which cause infectious hepatitis and laryngotracheitis in canid animals, respectively. They can be the backbones of viral vectors that could be applied in recombinant vaccines or for gene transfer in dogs and in serologically naïve humans. Although conventional plasmid-based reverse genetics systems can be used to construct CAdV vectors, their large genome size creates technical difficulties in gene cloning and manipulation. In this study, we established an improved reverse genetics system for CAdVs using bacterial artificial chromosomes (BACs), in which genetic modifications can be efficiently and simply made through BAC recombineering. To validate the utility of this system, we used it to generate CAdV2 with the early region 1 gene deleted. This mutant was robustly generated and attenuated in cell culture. The results suggest that our established BAC-based reverse genetics system for CAdVs would be a useful and powerful tool for basic and advanced practical studies with these viruses.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Establishing a foundation for large DNA transfer to artificial minichromosomes and B insert platforms in maize

2019 ◽  
Author(s):  
◽  
Jon Phalen Cody
Keyword(s):  
Complex Traits ◽  
Plant Cells ◽  
B Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Single Plant ◽  
Cloning Vectors ◽  
Artificial Chromosomes ◽  
Genetic Modifications ◽  
Independent Segregation

In plants, conventional genetic engineering methods limit the number of available traits that could potentially improve the quality of agriculture. Agrobacterium-mediated transformation and biolistic bombardment are tools used in transferring genes into plant cells, both of which result in random integrations into host genomes. The absence of targeting machinery, together with low DNA carrying capacity on most plasmid vectors, limit researchers to a few genes in a single modification experiment, a process that takes [about]1 year in most plant species. While stacking traits from independent genetic modifications allow for an increase in the number of transgenes in a single plant, recovery of all genes in subsequent generations becomes increasingly difficult due to independent segregation in meiosis. Alternatively, the use of binary bacterial artificial chromosomes (BiBACs), large insert cloning vectors, can maintain and transfer up to 300 kps, but are also subject to random integrations. Therefore, establishment of a BiBAC targeting system would be advantageous for researchers focusing on creating plant lines that contain several genes that work together to express complex traits, such as disease resistance clusters or whole biosynthetic pathways. Additionally, BiBAC targeting to a location outside the native chromosomal sets, such as an artificial minichromosome or B chromosome platform, would enable researchers to stack traits without disrupting endogenous sequences.

scholarly journals Patterns of genome size variation in snapping shrimp

Genome ◽  
2016 ◽  
Vol 59 (6) ◽  
pp. 393-402 ◽  
Author(s):  
Nicholas W. Jeffery ◽  
Kristin Hultgren ◽  
Solomon Tin Chi Chak ◽  
T. Ryan Gregory ◽  
Dustin R. Rubenstein
Keyword(s):  
Genome Size ◽  
Phylogenetic Signal ◽  
Size Variation ◽  
Diverse Group ◽  
Chromosome Size ◽  
Genome Size Variation ◽  
Large Genome ◽  
Large Genome Size ◽  
Single Genus ◽  
Geographic Regions

Although crustaceans vary extensively in genome size, little is known about how genome size may affect the ecology and evolution of species in this diverse group, in part due to the lack of large genome size datasets. Here we investigate interspecific, intraspecific, and intracolony variation in genome size in 39 species of Synalpheus shrimps, representing one of the largest genome size datasets for a single genus within crustaceans. We find that genome size ranges approximately 4-fold across Synalpheus with little phylogenetic signal, and is not related to body size. In a subset of these species, genome size is related to chromosome size, but not to chromosome number, suggesting that despite large genomes, these species are not polyploid. Interestingly, there appears to be 35% intraspecific genome size variation in Synalpheus idios among geographic regions, and up to 30% variation in Synalpheus duffyi genome size within the same colony.

Homologous Recombination Using Bacterial Artificial Chromosomes

2015 ◽  
Vol 2015 (2) ◽  
pp. pdb.prot072397
Author(s):  
Cary Lai ◽  
Tobias Fischer ◽  
Elizabeth Munroe
Keyword(s):  
Homologous Recombination ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication

2004 ◽  
Vol 78 (22) ◽  
pp. 12683-12688 ◽  
Author(s):  
Fernando Almazán ◽  
Carmen Galán ◽  
Luis Enjuanes
Keyword(s):  
Heterologous Protein ◽  
Cytopathic Effect ◽  
Human Cell Line ◽  
Genome Replication ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Transmissible Gastroenteritis ◽  
Transmissible Gastroenteritis Coronavirus

ABSTRACT The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

Applications of genetic markers in cytogenetic manipulation of the wheat genomes

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M. D. Gale ◽  
P. J. Sharp ◽  
S. Chao ◽  
C. N. Law
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Genetic Maps ◽  
Linkage Groups ◽  
Large Genome ◽  
Large Genome Size ◽  
Restriction Fragment Length

A molecular map of wheat, Triticum aestivum, is being developed. Problems associated with the large genome size, the large number of linkage groups, polyploidy, and limited polymorphism at the DNA level are being overcome. In addition to the breeding applications expected from the map, various uses for restriction fragment length polymorphism markers as tools in cytogenetic manipulation of wheat chromosomes and those from related species are being found. These include identification of aneuploid genotypes, added precision in intervarietal chromosome manipulations, tests of chromosome stability, identification of alien chromosomes, and marker-aided introgression of genes of agronomic importance from related species.Key words: wheat, restriction fragment length polymorphism, genetic maps, aneuploidy, alien chromosomes.

Use of Bacterial Artificial Chromosomes in Metagenomics Studies, Overview

2015 ◽  
pp. 671-680
Author(s):  
Lingling Wang ◽  
Shamima Nasrin ◽  
Mark Liles ◽  
Zhongtang Yu
Keyword(s):  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes
Sign in / Sign up

Export Citation Format

Share Document