Telomere-mediated chromosomal truncation in maize

Yu, Weichang; Lamb, Jonathan C.; Han, Fangpu; Birchler, James A.

doi:10.1073/pnas.0605750103

Cited by 111 publications

(146 citation statements)

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“…FISH screening of root tips of regenerated seedlings by using pWY96 (7) and pAHC25 probes separately revealed that both transgenes were present at the same locations in 84% of the transformants, as is often the case with cobombardment (14). There were 53 targeted B or miniB chromosomes that had both the pWY96 and pAHC25 transgenes (SI Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Chromosomal truncation was conducted by Agrobacterium-mediated transformation by using two telomere-containing constructs, pWY76 and pWY86, that possess a bialophos herbicide resistance selectable marker (bar), Cre/lox or FLP/FRT site-specific recombination cassettes, and the telomere repeats (7). During the integration event, the telomere-containing transgene caps the broken chromosome and seeds the production of a telomere.…”

Section: Resultsmentioning

confidence: 99%

“…Plasmid Construction and Gene Transformation. Telomere truncation constructs, pWY76 and pWY86, were reported previously (7). Plasmid pWY86-bar was prepared by digesting pWY86 with PmlI/AvrII and self-ligation to delete the 35S-bar gene expression cassette.…”

Section: Discussionmentioning

confidence: 99%

“…Agrobacterium-mediated transformation with pWY76 and pWY86 was performed as described (7). Biolistic-mediated gene transformation of maize immature embryos was conducted as described (42,43).…”

Section: Methodsmentioning

confidence: 99%

“…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-specific recombination cassettes for future manipulations.…”

mentioning

confidence: 99%

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Construction and behavior of engineered minichromosomes in maize

Han

Gao

et al. 2007

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

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136

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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: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%