Targefect-BAC mediated Efficient Transfection of Bacterial Artificial Chromosomes into mammalian cells

2018 ◽  
Author(s):  
rampyari walia ◽  
Rampyari Walia
Keyword(s):  
Mammalian Cells ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals Bacterial artificial chromosomes improve recombinant protein production in mammalian cells

2009 ◽  
Vol 9 (1) ◽  
pp. 3 ◽  
Author(s):  
Leander Blaas ◽  
Monica Musteanu ◽  
Robert Eferl ◽  
Anton Bauer ◽  
Emilio Casanova
Keyword(s):  
Recombinant Protein ◽  
Mammalian Cells ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells

BioTechniques ◽  
2003 ◽  
Vol 35 (4) ◽  
pp. 796-807 ◽  
Author(s):  
William J. Montigny ◽  
Stephanie F. Phelps ◽  
Sharon Illenye ◽  
Nicholas H. Heintz
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

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.

scholarly journals Isolation of Human Transcripts Expressed in Hamster Cells from YACs by cDNA Representational Difference Analysis

1999 ◽  
Vol 9 (2) ◽  
pp. 182-188
Author(s):  
Jessie Gu ◽  
Xin-Yuan Guan ◽  
Melissa A. Ashlock
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Positional Cloning ◽  
Yeast Artificial Chromosome ◽  
Expressed Sequence Tag ◽  
Gene Isolation ◽  
Representational Difference Analysis ◽  
Difference Analysis ◽  
Artificial Chromosomes ◽  
Link Type

Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA,NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF117641 and AF117642.]

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