Gene assembly in agrobacterium via nucleic acid transfer using recombinase technology (GAANTRY)

Authors: Hathwaik, Leyla; Thomson, James - Jim; Thilmony, Roger

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 9/8/2020
Publication Date: 1/21/2021
Citation: Hathwaik, L.T., Thomson, J.G., Thilmony, R.L. 2021. Gene assembly in agrobacterium via nucleic acid transfer using recombinase technology (GAANTRY). In: Bandyopadhyay, A., Thilmony, R.L, editors. Rice Genome Engineering and Gene Editing. Methods in Molecular Biology. New York, NY: Humana. p. 3-17.
DOI: https://doi.org/10.1007/978-1-0716-1068-8

Interpretive Summary: .

Technical Abstract: Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.