Rapid modification of bacterial artificial chromosomes by ET- recombination

Muyrers, Joep P P

doi:10.1093/nar/27.6.1555

Cited by 490 publications

(361 citation statements)

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“…These experiments confirm the early obervations of others (Zhang et al, 1998;Yu et al, 2000): recombineering can be as useful a tool for modifying plasmid replicons as it is for BACs Muyrers et al, 1999), the bacterial chromosome (Yu et al, 2000;Ellis et al, 2001;Costantino and Court, 2003;Thomason et al, 2005b), and phage λ itself (Oppenheim et al 2004), but there are complexities associated with multicopy plasmids that do not arise with the other replicons. ssDNA recombineering in the absence of mismatch repair targeted to plasmids is of such high efficiency that it can be used to isolate point mutations or changes of only a few bases without selection.…”

Section: Discussionsupporting

confidence: 74%

Multicopy plasmid modification with phage λ Red recombineering

Thomason¹,

Costantino²,

Shaw³

et al. 2007

Plasmid

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Recombineering, in vivo genetic engineering using the bacteriophage λ Red generalized recombination system, was used to create various modifications of a multicopy plasmid derived from pBR322. All genetic modifications possible on the E. coli chromosome and on bacterial artificial chromosomes (BACs) are also possible on multicopy plasmids and are obtained with similar frequencies to their chromosomal counterparts, including creation of point mutations (5-10% unselected frequency), deletions and substitutions. Parental and recombinant plasmids are nearly always present as a mixture following recombination, and circular multimeric plasmid molecules are often generated during the recombineering. Keywordsphage λ Red homologous recombination; multicopy plasmid; plasmid multimers; in vivo genetic engineering

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Section: Discussionsupporting

confidence: 74%

Multicopy plasmid modification with phage λ Red recombineering

Thomason¹,

Costantino²,

Shaw³

et al. 2007

Plasmid

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“…All three Bmp7 knockout alleles show very similar phenotypes in homozygosis, though some differences have been noted, particularly with respect to penetrance of the kidney al., 2003). The targeting vector was assembled on the BAC by two rounds of Bacterial Homologous Recombination (Muyrers et al, 1999) using generic modification cassettes shown in Fig. 1B.…”

Section: Resultsmentioning

confidence: 99%

Generation and functional characterization of mice with a conditional BMP7 allele

Zouvelou

Passa²,

Segklia³

et al. 2009

Int. J. Dev. Biol.

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Bone Morphogenetic Proteins (BMPs) play multiple and important roles in embryonic development as well as in homeostasis and tissue repair in the adult. Bmp7 has been implicated in developmental disorders and in a variety of diseases, but functional studies to elucidate its role so far have been hampered, since mice deficient in BMP7 die around or just after birth. To facilitate such studies, we generated mice in which the Bmp7 gene has been rendered conditional-null by flanking its first coding exon with loxP sites. To this end, we adapted the two-loxP site strategy to Bacterial Homologous Recombination to create a Bacterial Artificial Chromosome-based vector for direct targeting in mouse embryonic stem cells. Functional analysis showed that in vivo, the conditional-null Bmp7 flx/flx mice are phenotypically wild type, whereas post Cre-mediated recombination, the resulting Bmp7 ∆/∆ mice are phenotypically null. Thus, this study validates the usefulness of the Bmp7 flx/flx mouse which in turn should empower in vivo studies aimed at elucidating the roles of Bmp7 in postnatal development, homeostasis and disease.

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“…The simplicity of the coinjection strategy allows for efficient testing of multiple BACs for enhancer and rescue activity. More detailed studies then can be conducted by using homologous recombination in BACs (41,42) to delete or alter specific sequences to observe the effect on expression patterns. The BAC coinjection technique should facilitate the studies of control elements in many different genes.…”

Section: Discussionmentioning

confidence: 99%

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

DiLeone

Marcus

Johnson

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

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Rapid modification of bacterial artificial chromosomes by ET- recombination

Cited by 490 publications

References 0 publications

Multicopy plasmid modification with phage λ Red recombineering

Multicopy plasmid modification with phage λ Red recombineering

Generation and functional characterization of mice with a conditional BMP7 allele

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

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