INIR12 Transgenic Maize Marker Gene Excision
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Solution Overview
Problem
Current transgenic maize plants often contain undesirable rearrangements and selectable marker genes that are no longer needed, which can complicate genetic modification and trait expression, and existing methods for removing these markers are complex and require specific recombination site sequences.
Innovation Solution
Development of transgenic maize plant cells with specific nucleotide sequences lacking a second ZmUbilnt promoter and phosphomannose isomerase coding region, using gene editing molecules to introduce double-stranded DNA breaks and excise the INIR12 transgenic locus, allowing for the removal of unwanted DNA elements and marker genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If non-site specific integration methods are used to insert transgenes, then the process is simpler and more versatile, but undesirable rearrangements and multiple insertion sites occur
Solution Approach 1:
The transgene cassette is divided into separate functional modules: promoter, coding sequence, and terminator elements. This segmentation allows independent optimization of each component and enables precise control over expression patterns while maintaining the simplicity of the overall insertion process.
Solution Approach 2:
A selectable marker gene is introduced as an intermediary element linked to the transgene cassette. This marker serves as a mediator to identify and select plants with successful transgene integration, ensuring reliable expression while maintaining the simplicity of non-site specific integration methods.
2Ease of operation
If selectable marker genes are retained in transgenic plants, then selection and identification are easier, but genetic stability and trait expression are compromised
Solution Approach 1:
The patent removes unnecessary selectable marker genes from the final transgenic plant genome. By extracting these transiently useful markers after selection, the plant achieves improved genetic stability and cleaner trait expression while maintaining ease of operation during the selection process.
Solution Approach 2:
Selectable marker genes are used preliminarily during the transformation and selection process, then removed in subsequent generations. This preliminary use facilitates easy identification of transformed plants while their removal ensures long-term genetic stability and reliable trait expression.
3Manufacturing precision
If site-specific recombination systems are used to remove marker genes, then marker removal is more precise, but device complexity and requirement for specific recombination site sequences increase
Solution Approach 1:
The patent extracts and removes selectable marker genes from the transgenic plant genome through targeted deletion methods. This extraction approach achieves precise marker removal without requiring complex site-specific recombination systems, thereby maintaining manufacturing precision while reducing device complexity.
Solution Approach 2:
The patent uses molecular cloning and DNA sequencing techniques to create precise copies of the transgene cassette without marker genes. By working with purified DNA sequences, the method achieves precise marker removal and verification without the complexity of site-specific recombination systems.
Data Source
AI summary
Transgenic INIR12 maize plants comprising a vip3Aa19 expression cassette linked to a secondary nopaline synthase terminator element which lack a selectable marker gene are provided. In certain embodiments such INIR12 transgenic plants comprise modifications that provide for facile excision of the INIR12 transgenic locus from the maize plant genome. Genomic DNA of INIR12 transgenic plants, detection of INIR12 plants and products thereof, methods of making INIR12 plants, and use of INIR12 plants to facilitate breeding are disclosed.