Genome Manipulation Through Site-Specific Recombination

Ow, David W.; Medberry, Scott L.

doi:10.1080/713608117

Cited by 17 publications

(24 citation statements)

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“…Both constructs carry the gus reporter gene driven by a minimal CaMV 35S promoter on the mobile Ds, for enhancer trapping. Additionally, construct ET1 bears lox sequences for site-specific recombination (reviewed in Ow and Medberry 1995;Ow 1996) in the Ds transposon and the T-DNA. Upon crossing with lines expressing a Cre-recombinase, recombination can occur between two lox sites.…”

Section: Enhancer Trap Constructsmentioning

confidence: 99%

“…Some of the enhancer trap lines developed (construct ET1) bear lox sequences for site-specific recombination (reviewed in Ow and Medberry 1995;Ow 1996) in the Ds transposon and the T-DNA. Combining insertional mutagenesis with site-specific recombination provides additional advantages for mutant identification and gene cloning (Van Haaren and Ow 1993).…”

Section: Ds Transposition Behaviourmentioning

confidence: 99%

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Transpositional behaviour of an Ac/Ds system for reverse genetics in rice

Greco¹,

Ouwerkerk

Kam

et al. 2003

Theor Appl Genet

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A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.

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Section: Enhancer Trap Constructsmentioning

confidence: 99%

Section: Ds Transposition Behaviourmentioning

confidence: 99%

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice

Greco¹,

Ouwerkerk

Kam

et al. 2003

Theor Appl Genet

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“…The Cre/lox system is used to induce chromosomal inversions, translocations and deletions in planta (reviewed by Ow and Medberry (1995) and Gilbertson (2003)). …”

Section: Introductionmentioning

confidence: 99%

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

et al. 2005

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Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.

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“…Recombination events mediated by the Cre recombinase lead to the excision or the inversion of DNA sequences located between two direct or inverted repeats of loxP sites, respectively. In plants, the Cre/lox system was successfully used to generate mutations as diverse as translocations, deletions, or inversions and to regulate conditional gene expression or site-specific integration (83,84,126). One of its direct applications in plant biotechnology is the excision of selectable markers and plasmid sequences in genetically modified crops (31,133).…”

Section: Cre/lox-mediated Site-specific Recombination In Physcomitrellamentioning

confidence: 99%

A NEWMOSSGENETICS: Targeted Mutagenesis inPhyscomitrella patens

Schaefer

2002

Annu. Rev. Plant Biol.

162

110

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s Abstract The potential of moss as a model system to study plant biology is associated with their relatively simple developmental pattern that nevertheless resembles the basic organization of the body plan of land plants, the direct access to cell-lineage analysis, their similar responses to plant growth factors and environmental stimuli as those observed in other land plants, and the dominance of the gametophyte in the life cycle that facilitates genetic approaches. Transformation studies in the moss Physcomitrella patens have revealed a totally unique feature for plants, i.e., that foreign DNA sequences integrate in the genome preferentially at targeted locations by homologous recombination, enabling for the first time in plants the application of the powerful molecular genetic approaches used routinely in bacteria, yeast, and since 1989, the mouse embryonic stem cells. This article reviews our current knowledge of Physcomitrella patens transformation and its unique suitability for functional genomic studies.

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Genome Manipulation Through Site-Specific Recombination

Cited by 17 publications

References 0 publications

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

A NEWMOSSGENETICS: Targeted Mutagenesis inPhyscomitrella patens

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