Repositorium

What is a repositorium?

The repositorium is a searchable database that provides data on relevant articles from journals, company web pages and web pages of governmental agencies about studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Search options are article type, technique, plant, traits or free text. The repositorium is based on the systematic map of Dominik Modrzejewski et al., published in the journal environmental evidence. (Download article PDF).

Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion


Typ / Jahr

Journal Article / 2015

Autoren

Nandy, Soumen; Zhao, Shan; Pathak, Bhuvan P.; Manoharan, Muthusamy; Srivastava, Vibha

Abstract

BACKGROUND Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. METHODS All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. RESULTS We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cre-mediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. CONCLUSIONS A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites.

Keywords
Biotechnology; Cre-lox; Deoxyribonucleases/genetics; FLP-FRT; gene deletion; Gene stacking; Genetic Engineering/methods; Genetic Markers/genetics; Genetic Vectors/genetics; Integrases/genetics/metabolism; I-SceI; Multigene transformation; Oryza/genetics; Plants, Genetically Modified/genetics; Site-specific recombination; Targeted gene integration; ZFN
Periodical
BMC biotechnology
Periodical Number
Page range
93
Volume
15
DOI
10.1186/s12896-015-0212-2

Techniques

ID Corresponding Author
Country
Plant Species GE Technique
Sequence Identifier
Trait
Type of Alteration
Progress in Research
Key Topic
330 Srivastava, Vibha
USA
Oryza sativa Meganucleases
I-SCE-I marker flanking bar gene
No information
SDN1
Basic research
Basic research
331 Srivastava, Vibha
USA
Oryza sativa Zinc-finger nucleases
CCR5
No information
SDN1
Basic research
Basic research