Gene targeting approaches using positive-negative selection and large flanking regions

Area of Science:

• Plant molecular biology
• Genetics
• Biotechnology

Background:

• Gene targeting is crucial for plant genetic engineering.
• Efficient selection of targeted events is a major challenge.
• Previous methods often suffer from low efficiency and high background noise.

Purpose of the Study:

• To enhance the frequency of detectable gene targeting in plants.
• To improve the selection efficiency of double-recombinants in transgenic calli.
• To optimize gene targeting strategies in Lotus japonicus.

Main Methods:

• Utilized Agrobacterium tumefaciens T-DNA replacement vectors for gene targeting at Gln1 and Pzfloci in Lotus japonicus.
• Employed large flanking regions (up to 22.9 kb) for homologous recombination.
• Integrated a negative selectable marker (cytosine deaminase) alongside a positive selection marker for double-recombinant selection.

Main Results:

• Achieved over 1000-fold enrichment of putative targeting events using negative selection, demonstrating its efficiency.
• Selected 185 resistant calli from 18,974 transformation events using five replacement vectors.
• Failed to verify gene targeting events in survivors via PCR screening and Southern blot analysis.

Conclusions:

• The combined positive and negative selection strategy significantly enriched for potential gene targeting events.
• Despite optimization, the frequency of detectable gene targeting in Lotus japonicus remained below 5.3 x 10(-5).
• Further improvements in selection or targeting mechanisms are needed for practical plant gene editing.