Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors

Abstract

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-loxcre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

Chromosomes with a life of their own

“…Birchler, unpublished work). Agrobacterium -mediated transformation was first used and transgene inserts were localized among all 10 A chromosomes but no inserts were found on the Bs; thus a bias against the maize B by the Agrobacterium -mediated transformation was revealed (Vega et al, 2008). Yu et al (2007) then used biolistic-mediated transformation with telomere-containing plasmids and obtained B chromosomes truncated at different positions.…”

Telomere-mediated truncation of barley chromosomes

“…A comparable telomere seeding strategy was used for the generation of truncated chromosomes in the frog Xenopus laevis (Wuebbles and Jones 2007), the fungus Candida albicans (Kabir and Rustchenko 2005) and the protozoon Leishmania (Tamar and Papadopoulou 2001). Yu et al (2006) and Vega et al(2008) successfully adapted telomere seeding for the formation of truncated chromosomes in plants. T-DNA constructs containing arrays of Arabidopsis-type telomere repeats (Richards and Ausubel 1988) were transferred into maize immature embryos to generate transgenic lines carrying chromosomes reduced in size.…”

“…However, T-DNA-induced chromosome truncation additionally provides a platform for targeted integration of expression cassettes and for stabilisation of truncated chromosomes by telomere seeding. Subsequent reporter gene transfer from a terminal transgene on an otherwise normal A chromosome of maize to an engineered minichromosome was achieved by means of Cre/lox-based site-specific recombination (Vega et al 2008;Yu et al 2007). Because targeting of transgenes to particular genomic sequences by homologous recombination is inefficient in seed plants (Reiss 2003), a targeted delivery of constructs containing telomere repeats to particular chromosome sites as used in mammals (Lim and Farr 2004) is not yet possible.…”

Minichromosomes: Vectors for Crop Improvement

“…Shortly after, the vectors were modified to have the ability to be maintained in Agrobacterium tumefaciens as transformation vectors [50]. The result, known as binary bacterial artificial chromosome (BIBAC) vectors, have been shown to transfer large intact DNA fragments in a variety of different plant species [50][51][52]. By including telomere sequences near the right border of a BIBAC vector, it should be possible to create minichromosomes while introducing large DNA fragments.…”