Site‐specific integration of DNA into wild‐type and mutant <i>lox</i> sites placed in the plant genome

Albert, Henrik H.; Dale, Emily C.; Lee, Elsa; Ow, David W.

doi:10.1046/j.1365-313x.1995.7040649.x

Cited by 447 publications

(441 citation statements)

References 28 publications

(34 reference statements)

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“…The J11-9 parent supplies the Cre recombinase and an alternative lox recombination site for introducing the 35S promoter to activate the DsRed gene. The recombination target sites, lox66 and lox71, are mutated lox sequences that can recombine most favorably in the forward reaction (26). Successful exchange of the two transgenes will result in the transfer of the distal regions of the two transgenes, produce a red fluorescence protein by placing the promoterless DsRed gene under the control of the 35S promoter, and the addition of genetic material to the minichromosome from J11-9 (Fig.…”

Section: Recombination Of Minichromosomesmentioning

confidence: 99%

Construction and behavior of engineered minichromosomes in maize

Han

Gao

et al. 2007

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

142

138

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Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.artificial chromosomes ͉ FISH ͉ genetic engineering ͉ telomere truncation A rtificial chromosomes involving de novo centromere formation on an independently assembled unit and engineered minichromosomes produced by telomere truncation provide striking advantages over traditional methods of gene transformation in yeast and mammalian cells (1-5). The development of such chromosomes in plants would provide these advantages for many applications in basic studies, biotechnology, and agriculture. These chromosomes could be used as independent platforms for foreign gene expression without random integration into the normal chromosomes. Further additions of unlimited amounts of DNA could be added to these platforms in a sequential manner via different site-specific recombination cassettes. Genes introduced in this way would be present in a defined context and thus could be expressed at a more predictable level than through random integration (6). Hence, additional genes, multigene complexes, or even whole metabolic pathways could potentially be added to a genotype. Moreover, engineered or artificial chromosomes could be easily introduced or removed from a genotype by genetic crosses and would facilitate introgression of transgenes to different genetic backgrounds.To extend engineered chromosome technology to plants, we developed a method of telomere-mediated chromosomal truncation in maize by Agrobacterium-mediated transformation of constructs with multiple copies of the telomere sequence (7). Here, we report the use of this technology to produce minichromosome platforms by truncating both normal A and supernumerary B maize chromosomes and at the same time introducing site-spe...

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Section: Recombination Of Minichromosomesmentioning

confidence: 99%

Construction and behavior of engineered minichromosomes in maize

Han

Gao

et al. 2007

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

142

138

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“…Probes located 5 0 and 3 0 of the respective Mll fragment (Mll 5 0 probe, Mll 3 0 probe). The invertor cassette is depicted below the Mll map and two forms were used, one flanked by wild-type loxP sequences (loxP) and a second generation by the mutant loxP sites (lox66 and lox71 (Albert et al, 1995)). These sequences are shown below the AF4 invertor cassette.…”

Section: Mll-af4 Fusion Is Lethal For Mouse Embryosmentioning

confidence: 99%

“…This Mll-AF4 knock-in model was not subject to embryonic lethality, allowing inter-breeding with lines of mice expressing Cre recombinase in various specific haematopoietic cell types. Two sets of Mll-AF4 invertor ES cells were made in which the AF4 cassette was flanked by either wild-type or mutant loxP sites (Hoess et al, 1982;Albert et al, 1995) (Mll-AF4 loxP and Mll-AF4 lox66/71 invertors, Figure 1a). Following expression of Cre recombinase in the ES cells, the invertor cassette knocked-in to Mll intron 10 ( Figure 1b) became inverted and thus was orientated in the same transcriptional direction to Mll (Figure 1c) and was transcribed into a fusion mRNA of Mll-AF4 (Figure 1d and e).…”

Section: Mll-af4 Invertor Mouse Model M Metzler Et Almentioning

confidence: 99%

A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology

et al. 2006

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MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukaemia and is associated with a poor prognosis. MLL-AF4 is thought to be required in haematopoietic stem cells to elicit leukaemia and may be involved in tumour phenotype specification as it is only found in B-cell tumours in humans. We have employed the invertor conditional technology to create a model of MLL-AF4, in which a floxed AF4 cDNA was knocked into Mll in the opposite orientation for transcription. Cell-specific Cre expression was used to generate Mll-AF4 expression. The mice develop exclusively B-cell lineage neoplasias, whether the Cre gene was controlled by B-or T-cell promoters, but of a more mature phenotype than normally observed in childhood leukaemia. These findings show that the MLL-AF4 fusion protein does not have a mandatory role in multi-potent haematopoietic stem cells to cause cancer and indicates that MLL-AF4 has an instructive function in the phenotype of the tumour.

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“…First, an excess amount of Cre may have a cytotoxic effect on cell growth (Baba et al, 2005). Second, the excision reaction may be promoted for mutated loxPs with a double arm mutation in the presence of excess amounts of Cre, although an irreversible integration reaction can be achieved by poor recognition for loxPs with a double arm mutation (Albert et al, 1995). From the fact that the subsequent target gene could be accumulatively integrated into chromosome without any deletion of the previously integrated genes both in vitro and on the CHO genome, at least, we could assume that the recombination between loxPs having different spacers was not occurred.…”

Section: Discussionmentioning

confidence: 99%

An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

Kameyama

Kawabe

Ito

et al. 2010

Biotech & Bioengineering

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The Cre-loxP system is frequently used for sitespecific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms.

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Site‐specific integration of DNA into wild‐type and mutant lox sites placed in the plant genome

Cited by 447 publications

References 28 publications

Construction and behavior of engineered minichromosomes in maize

Construction and behavior of engineered minichromosomes in maize

A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology

An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

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