scholarly journals Plasmid Production

2020 ◽  
Author(s):  
Keyword(s):  
Plasmid Production

scholarly journals Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors

2019 ◽  
Vol 116 (52) ◽  
pp. 27001-27010 ◽  
Author(s):  
Kyle B. Fischer ◽  
Hannah K. Collins ◽  
Edward M. Callaway
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Transgene Expression ◽  
Cell Types ◽  
Cell Type ◽  
Recognition Sites ◽  
Highly Sensitive ◽  
Plasmid Production ◽  
Targeted Gene Expression ◽  
Mouse Lines

In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or “leak” expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant “cross-over insensitive” recognition sites combined with an “ATG-out” design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.

Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol

2004 ◽  
Vol 6 (9) ◽  
pp. 963-973 ◽  
Author(s):  
Xiaobin Lu ◽  
Laurent Humeau ◽  
Vladimir Slepushkin ◽  
Gwendolyn Binder ◽  
Qiao Yu ◽  
...  
Keyword(s):  
Primary Cells ◽  
Lentivirus Vector ◽  
One Step ◽  
Plasmid Production

In situNIR spectroscopy monitoring of plasmid production processes: effect of producing strain, medium composition and the cultivation strategy

2014 ◽  
Vol 90 (2) ◽  
pp. 255-261 ◽  
Author(s):  
Marta B. Lopes ◽  
Geisa A. L. Gonçalves ◽  
Daniel Felício-Silva ◽  
Kristala L. J. Prather ◽  
Gabriel A. Monteiro ◽  
...  
Keyword(s):  
Medium Composition ◽  
Production Processes ◽  
Plasmid Production ◽  
Cultivation Strategy

Mini-scale cultivation method enables expeditious plasmid production inEscherichia coli

2013 ◽  
Vol 9 (1) ◽  
pp. 128-136 ◽  
Author(s):  
Petra Grunzel ◽  
Maciej Pilarek ◽  
Dörte Steinbrück ◽  
Antje Neubauer ◽  
Eva Brand ◽  
...  
Keyword(s):  
Inescherichia Coli ◽  
Cultivation Method ◽  
Plasmid Production

scholarly journals Production of Two Proteins Encoded by theBacteroides Mobilizable Transposon NBU1 Correlates with Time-Dependent Accumulation of the Excised NBU1 Circular Form

2001 ◽  
Vol 183 (21) ◽  
pp. 6335-6343 ◽  
Author(s):  
Jun Wang ◽  
Gui-Rong Wang ◽  
Nadja B. Shoemaker ◽  
Abigail A. Salyers
Keyword(s):  
Gene Dosage ◽  
Constitutive Expression ◽  
The Other ◽  
Time Dependent ◽  
Multicopy Plasmid ◽  
Late Exponential Phase ◽  
Western Blots ◽  
Circular Form ◽  
Genes Encoding ◽  
Plasmid Production

ABSTRACT NBU1 is a mobilizable transposon that excises from theBacteroides chromosome to form a double-stranded circular transfer intermediate. Excision is triggered by exposure of the bacteria to tetracycline. Accordingly, we expected that the expression of NBU1 genes would be induced by tetracycline. To test this hypothesis, antibodies that recognized two NBU1-encoded proteins, PrmN1 and MobN1, were used to monitor production of these proteins. PrmN1 is essential for excision, and MobN1 is essential for transfer of the excised circular form. At first, expression of the genes encoding these two proteins appeared to be regulated by tetracycline, because the proteins were detectable on Western blots only after the cells were exposed to tetracycline. However, when the prmN1 gene and/or the mobN1 gene was cloned on a multicopy plasmid, production of the protein was constitutive. Initially, we assumed that the constitutive expression was due to loss of a repressor protein that was encoded by one of the other genes on NBU1. Deletions or insertions in the other genes (orf2 and orf3) on NBU1 and various integrated NBU1 derivatives abolished production of PrmN1 and MobN1. This is the opposite of what should have happened if one or both of these genes encoded a repressor. A second possibility was that when NBU1 excised, it replicated transiently, increasing the gene dosage of prmN1 and mobN1 and thereby producing enough PrmN1 and MobN1 for these proteins to become detectable. In fact, after the cells entered late exponential phase the copy number of NBU1 increased to 2 to 3 copies per cell. Production of PrmN1 and MobN1 showed a similar pattern. Any mutation in NBU1 that decreased or prevented excision also prevented elevated production of these two proteins. Our results show that the apparent tetracycline dependence of the production of PrmN1 and MobN1 is due to a growth phase- or time-dependent increase in the number of copies of the NBU1 circular form.

Mitochondrial Gene Therapy: Advances in Mitochondrial Gene Cloning, Plasmid Production, and Nanosystems Targeted to Mitochondria

2017 ◽  
Vol 14 (3) ◽  
pp. 626-638 ◽  
Author(s):  
Eduarda Coutinho ◽  
Cátia Batista ◽  
Fani Sousa ◽  
João Queiroz ◽  
Diana Costa
Keyword(s):  
Gene Therapy ◽  
Gene Cloning ◽  
Mitochondrial Gene ◽  
Plasmid Production
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