Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination

Noll, Stephan; Hampp, Gabriele; Bausbacher, Hanna; Pellegata, Natalia S.; Kranz, Harald

doi:10.2144/000113150

Cited by 9 publications

(6 citation statements)

References 23 publications

(16 reference statements)

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“…Plasmid recombineering (Noll et al ., ) was used for the generation of the cckA deletion construct. Primers cckA‐p1 and cckA‐p2 (Table ) were used to amplify a 4351‐bp region encoding cckA ( rcc01749 ) plus ~1 kb of flanking sequence on each side.…”

Section: Methodsmentioning

confidence: 99%

Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus

Mercer

Quinlan

Rose

et al. 2012

FEMS Microbiol Lett

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Production of the gene transfer agent of Rhodobacter capsulatus, RcGTA, is dependent upon several cellular regulatory systems, including a putative phosphorelay involving the CtrA and CckA proteins. These proteins are also involved in flagellar motility in R. capsulatus. The interactions of proteins in this system are best understood in Caulobacter crescentus where CtrA is activated by phosphorylation by the CckA‐ChpT phosphorelay. CtrA~P activity is further controlled by SciP, which represses ctrA transcription and CtrA activation of transcription. We show that R. capsulatus chpT and cckA mutants both have greatly reduced motility and RcGTA activity. Unlike the ctrA mutant where RcGTA gene transcription is absent, the decrease in RcGTA activity is because of reduced release of RcGTA from the cells. The sciP mutant is not affected for RcGTA production but our results support the C. crescentus model of SciP repression of flagellar motility genes. We show that both unphosphorylated and phosphorylated CtrA can activate RcGTA gene expression, while CtrA~P seems to be required for release of the particle and expression of motility genes. This has led us to a new model of how this regulatory system controls motility and production of RcGTA in R. capsulatus.

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Section: Methodsmentioning

confidence: 99%

Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus

Mercer

Quinlan

Rose

et al. 2012

FEMS Microbiol Lett

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“…Neuroligins (NLGNs) are a family of synaptic adhesion molecules highly expressed in the brain and are ligands for another group of cell adhesion molecules neurexins (NRXNs) (Ichtchenko et al, 1995). There are five neuroligin genes (neuroligin-1, -2, -3, -4, and -5) in humans and four in mice (neuroligin [1][2][3][4]. Neuroligin-1, -2, and -3 are close homologs between human and mice.…”

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confidence: 99%

GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation

Chen

Jiang

et al. 2017

Preprint

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Schizophrenia (SCZ) is a chronic neuropsychiatric disorder caused by both genetic and environmental factors. It is featured by long-standing delusion and hallucination (psychosis), and cognitive deficits (Freedman, 2003; Insel, 2010; Lewis and Lieberman, 2000). SCZ is a highly heritable disorder (Sullivan et al., 2003) with a complex genetic basis. Recent genomic studies identified a number of genetic variants associated with . CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/225524 doi: bioRxiv preprint first posted online Nov. 27, 2017; 3 SCZ, including a group of variants resided in the genes encoding synaptic adhesion molecules that promoting synaptic function and development such as IGSF9B, and NLGN4X (Schizophrenia Working Group of the Psychiatric Genomics, 2014).Neuroligins (NLGNs) are a family of synaptic adhesion molecules highly expressed in the brain and are ligands for another group of cell adhesion molecules neurexins (NRXNs) (Ichtchenko et al., 1995). There are five neuroligin genes (neuroligin-1, -2, -3, -4, and -5) in humans and four in mice (neuroligin 1-4). Neuroligin-1, -2, and -3 are close homologs between human and mice. Neuroligin-1 and neuroligin-2 differentially locate to excitatory and inhibitory synapses and are critical for the excitatory and inhibitory synapse formation and function, respectively (Chubykin et al., 2007;Dong et al., 2007; Levinson et al., 2005; Nam and Chen, 2005;Scheiffele et al., 2000;Song et al., 1999;Varoqueaux et al., 2004). Neuroligin-3 locates at both type of synapses and contributes to both neurotransmission (Budreck and Scheiffele, 2007; Etherton et al., 2011). In recent years, several genetic variants of neuroligin-3 and neuroligin-4 have been identified in autism patients (Chih et al., 2004; Jamain et al., 2003). Mutations in proteins interacting with neuroligins such as Neurexin1, SHANK and MDGA have also been associated with autism and schizophrenia patients (Bucan et al., 2009; Durand et al., 2007; Kim et al., 2008; Kirov et al., 2008). Genetic mouse models based on these findings recapitulate some aspects of patient symptoms as well (Baudouin et al., 2012;Connor et al., 2016; Etherton et al., 2011; Etherton et al., 2009; Jamain et al., 2008;Peça et al., 2011;Rothwell et al., 2014;Südhof, 2008;Tabuchi et al., 2007; Zhou et al., 2016).. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/225524 doi: bioRxiv preprint first posted online Nov. 27, 2017; 4We have previously reported several novel mutations of NLGN2 from schizophrenia patients (Sun et al., 2011). Among the NL2 mutants, we found that the R215H mutant protein was retained in the endoplasmic...

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“…It is therefore possible to insert functional elements at virtually every position in a large plasmid. Typically, Red/ET recombination occurs between a linear and a circular reaction partner using homology arms of approximately 50 bp in length (7), which are conveniently attached to the linear reaction partner by polymerase chain reaction (PCR).…”

Section: Methodsmentioning

confidence: 99%

Lentiviral-Based Approach for the Validation of Cancer Therapeutic Targets in Vivo

Ambrogio

Stern²,

Scuoppo

et al. 2014

BioTechniques

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Despite the pressing need for novel cancer treatments, our improved understanding of tumor biology is not being successfully translated into better therapies. Here we present a lentiviral vector that enables in vivo validation of cancer therapeutic targets when combined with existing cancer animal models that faithfully reproduce the natural history of human disease. Unlike the conventional genetic approaches with targeted alleles, the outlined experimental strategy could be used to assess the preclinical efficacy of a growing number of putative therapeutic hits in a rapid and cost-effective manner.

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Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination

Cited by 9 publications

References 23 publications

Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus

Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus

GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation

Lentiviral-Based Approach for the Validation of Cancer Therapeutic Targets in Vivo

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