The Flp double cross system a simple efficient procedure for cloning DNA fragments

Sadowski, Paul D.

doi:10.1186/1472-6750-3-9

Cited by 9 publications

(1 citation statement)

References 25 publications

(26 reference statements)

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“…In order to use EcNR2 as production host cells following MAGE, the inactivated MMR system should be rescued. In the case of EcSIM strains, the suicide plasmids (pRED plasmids) can effectively be excised from their chromosomal insertion sites through a second recombination event [27] , [28] , allowing for only transient inactivation of the MMR system during the genome engineering process.…”

Section: Resultsmentioning

confidence: 99%

A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering

et al. 2014

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Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.

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Section: Resultsmentioning

confidence: 99%

A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering

et al. 2014

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Maize and the Biotech Industry

Johnson

McCuddin

Handbook of Maize

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Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression

Real

Monteiro

Burger

et al. 2011

Appl Microbiol Biotechnol

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Lentiviral vectors are an important tool for gene delivery in vivo and in vitro. The success of gene transfer approaches relies on high and stable levels of gene expression. To this end, several molecular strategies have been employed to manipulate these vectors towards improving gene expression in the targeted animal cells. Low gene expression can be accepted due to the weak transcription from the majority of available mammalian promoters; however, this obstacle can be in part overcome by the insertion of cis-acting elements that enhance gene expression in various expression contexts. In this work, we created different lentiviral vectors in which several posttranscriptional regulatory elements, namely the Woodchuck hepatitis posttranscriptional regulatory element (WPRE) and different specialized poly(A) termination sequences (BGH and SV40) were used to develop vectors leading to improved transgene expression. These vectors combine the advantages of restriction enzyme/ligation-independent cloning eliminating the instability and recombinogenic problems occurring from traditional cloning methods in lentiviral expression vectors and were tested by expressing GFP and the firefly Luciferase reporter gene from different cellular promoters in different cell lines. We show that the promoter activity varies between cell lines and is affected by the lentiviral genomic context. Moreover, we show that the combination of the WPRE element with the BGH poly(A) signal significantly enhances transgene expression. The vectors herein created can be easily modified and adapted without the need for extensive recloning making them a valuable tool for viral vector development.

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The Flp double cross system a simple efficient procedure for cloning DNA fragments

Cited by 9 publications

References 25 publications

A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering

A Simple and Effective Method for Construction of Escherichia coli Strains Proficient for Genome Engineering

Maize and the Biotech Industry

Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression

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