Construction of Gene-Targeting Vectors by Recombineering

Lee, Song Choon

doi:10.1101/pdb.prot5291

Cited by 9 publications

(4 citation statements)

References 26 publications

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“…The knockout construct was generated by recombineering (Lee and Liu, 2009), linearized, and electroporated into C2 ESCs (Gertsenstein et al., 2010). Correctly targeted clones were verified by both genomic PCR and Southern blot, and injected into C57BL/6 blastocysts.…”

Section: Methodsmentioning

confidence: 99%

CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State

et al. 2016

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SummaryPoly(A) tail length and mRNA deadenylation play important roles in gene regulation. However, how they regulate embryonic development and pluripotent cell fate is not fully understood. Here we present evidence that CNOT3-dependent mRNA deadenylation governs the pluripotent state. We show that CNOT3, a component of the Ccr4-Not deadenylase complex, is required for mouse epiblast maintenance. It is highly expressed in blastocysts and its deletion leads to peri-implantation lethality. The epiblast cells in Cnot3 deletion embryos are quickly lost during diapause and fail to outgrow in culture. Mechanistically, CNOT3 C terminus is required for its interaction with the complex and its function in embryonic stem cells (ESCs). Furthermore, Cnot3 deletion results in increases in the poly(A) tail lengths, half-lives, and steady-state levels of differentiation gene mRNAs. The half-lives of CNOT3 target mRNAs are shorter in ESCs and become longer during normal differentiation. Together, we propose that CNOT3 maintains the pluripotent state by promoting differentiation gene mRNA deadenylation and degradation, and we identify poly(A) tail-length regulation as a post-transcriptional mechanism that controls pluripotency.

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Section: Methodsmentioning

confidence: 99%

CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State

et al. 2016

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“…More recently, other targeting designs have been described that require an exact knowledge of the sequence of the chosen integration region and are performed knocking down selectable marker genes (21,37). A promising improvement in gene targeting has been recently achieved by means of "recombineering," a technique that utilizes the homologous recombination functions encoded by gamma phage to construct knockout vectors (reviewed in (38)). …”

Section: Size Reduction Of Natural Minichromosomesmentioning

confidence: 99%

Naturally Occurring Minichromosome Platforms in Chromosome Engineering: An Overview

Raimondi

2011

Methods in Molecular Biology

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Artificially modified chromosome vectors are non-integrating gene delivery platforms that can shuttle very large DNA fragments in various recipient cells: theoretically, no size limit exists for the chromosome segments that an engineered minichromosome can accommodate. Therefore, genetically manipulated chromosomes might be potentially ideal vector systems, especially when the complexity of higher eukaryotic genes is concerned. This review focuses on those chromosome vectors generated using spontaneously occurring small markers as starting material. The definition and manipulation of the centromere domain is one of the main obstacles in chromosome engineering: naturally occurring minichromosomes, due to their inherent small size, were helpful in defining some aspects of centromere function. In addition, several distinctive features of small marker chromosomes, like their appearance as supernumerary elements in otherwise normal karyotypes, have been successfully exploited to use them as gene delivery vectors. The key technologies employed for minichromosome engineering are: size reduction, gene targeting, and vector delivery in various recipient cells. In spite of the significant advances that have been recently achieved in all these fields, several unsolved problems limit the potential of artificially modified chromosomes. Still, these vector systems have been exploited in a number of applications where the investigation of the controlled expression of large DNA segments is needed. A typical example is the analysis of genes whose expression strictly depends on the chromosomal environment in which they are positioned, where engineered chromosomes can be envisaged as epigenetically regulated expression systems. A novel and exciting advance concerns the use of engineered minichromosomes to study the organization and dynamics of local chromatin structures.

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“…Due to the strength of this promoter, 15 minutes was ample time for recombination to occur, and approximately 1 in 500 clones carried the desired mutation [ 69 ]. Because recombination functions are expressed under the control of the natural promoter, this system is very efficient, tightly regulated and genes are expressed in their physiological molar ratio [ 79 ]. This temperature-sensitive repressor system was also constructed on a non-replicating circular plasmid termed mini-λ [ 80 ].…”

Section: Bacterial Genomic Recombinationmentioning

confidence: 99%

“…This temperature-sensitive repressor system was also constructed on a non-replicating circular plasmid termed mini-λ [ 80 ]. A range of other conditional plasmids, both high and low copy number, carrying recombination functions have also been used, similar to those described above for RecA-dependent recombination [ 79 , 81 , 82 ]. The use of plasmid constructs to provide recombination functions limits the range of plasmids that can be used for subsequent manipulations [ 69 ].…”

Section: Bacterial Genomic Recombinationmentioning

confidence: 99%

Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

Hall

Meers

Fowler³

et al. 2012

Viruses

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Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.

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Construction of Gene-Targeting Vectors by Recombineering

Cited by 9 publications

References 26 publications

CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State

CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State

Naturally Occurring Minichromosome Platforms in Chromosome Engineering: An Overview

Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

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