scholarly journals Highly Efficient Modification of Bacterial Artificial Chromosomes (BACs) Using Novel Shuttle Vectors Containing the R6Kgamma Origin of Replication

2002 ◽  
Vol 12 (12) ◽  
pp. 1992-1998 ◽  
Author(s):  
S. Gong
Keyword(s):  
Origin Of Replication ◽  
Bacterial Artificial Chromosomes ◽  
Shuttle Vectors ◽  
Artificial Chromosomes ◽  
Highly Efficient

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.

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.

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

Spiroplasmas as Biological Control Agents of Insect Pests

1995 ◽  
Author(s):  
Kevin Hackett ◽  
Shlomo Rottem ◽  
David L. Williamson ◽  
Meir Klein
Keyword(s):  
Leptinotarsa Decemlineata ◽  
Virulence Genes ◽  
Insect Pests ◽  
The United States ◽  
Origin Of Replication ◽  
Shuttle Vectors ◽  
Large Unilamellar Vesicles ◽  
Extrachromosomal Elements ◽  
Genetically Engineer ◽  
Large Clusters

Toward development of spiroplasmas as novel toxin-delivery systems for biocontrol of beetle pests in the United States (Leptinotarsa decemlineata) and Israel (Maladera matrida), media for cultivating beetle-associated spiroplasmas were improved and surveys of these spiroplasmas were conducted to provide transformable strains. Extensive surveys of spiroplasmas yielded promising extrachromosomal elements for vector constructs. One, plasmid pCT-1, was cloned, characterized, and used as a source of spiroplasma origin of replication in our shuttle vectors. The fibrillin gene was isolated and sequenced and its strong promoter was also used in the constructs. Means for transforming these vectors into spiroplasmas were developed and optimized, with electroporation found to be suitable for most applications. Development and optimization of means for using large unilamellar vesicles (LUVs) in spiroplasma transformation represents a breakthrough that should facilitate insertion of large clusters of virulence genes. With completion of the vector, we should thus be poised to genetically engineer spiroplasmas with genes that will express toxins lethal to our target beetles, thus providing an effective and inexpensive alternative to conventional means of beetle control.

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