Direct Protein Introduction for Eukaryotic Genome Rearrangement
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods for breeding eukaryotes using double-stranded DNA restriction enzymes require multiple steps, stress plants with heat treatment, and can lead to unanticipated genetic recombination and difficulty in genetic separation, especially in vegetatively propagating plants with poor seed-forming ability.
Innovation Solution
Introducing a protein with double-stranded DNA cleavage activity directly into eukaryotes to induce genetic recombination and rearrangement, avoiding the need for heat activation and exogenous gene expression, thereby simplifying the breeding process and stabilizing traits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genes coding for restriction enzymes are introduced into eukaryotes for genome rearrangement, then genetic recombination can be induced, but the process requires multiple passages (5-6 passages) to obtain stable traits and involves time-consuming gene transcription and translation
Solution Approach 1:
The patent extracts the restriction enzyme protein from the eukaryotic cell system by introducing it directly as a purified protein rather than as a gene that requires transcription and translation. This eliminates the time-consuming gene expression process and reduces the number of passages needed to achieve genome rearrangement and stable trait acquisition.
Solution Approach 2:
The restriction enzyme is prepared in advance as a purified protein product before introduction into the eukaryote. This preliminary preparation of the active enzyme form bypasses the need for in-cell gene expression, allowing immediate action on the genome upon introduction and significantly reducing the time required for breeding operations.
2Reliability
If heat treatment is applied to activate restriction enzyme for genome rearrangement, then DNA cleavage activity is enhanced, but plant growth and yields are adversely affected due to heat stress
Solution Approach 1:
The patent changes the activation parameter from temperature (heat treatment) to protein concentration and specificity. By using restriction enzymes with recognized specific base sequences and introducing them at appropriate concentrations, the DNA cleavage activity is reliably activated without applying heat stress that would harm plant growth and yields.
Solution Approach 2:
The patent converts the potential harm of heat treatment into a benefit by selecting restriction enzymes that are highly specific and active at physiological temperatures. The specificity of these enzymes allows reliable DNA cleavage without the need for heat activation, thereby eliminating heat stress while maintaining effective genome rearrangement capability.
3Reliability
If restriction enzyme is expressed continuously in eukaryote to maintain DNA cleavage activity, then genome rearrangement can occur, but unanticipated genetic recombination occurs and growth and survival are adversely affected
Solution Approach 1:
The patent implements periodic rather than continuous action by introducing the restriction enzyme as a transient protein expression system. The enzyme is introduced at specific time points to perform DNA cleavage and genome rearrangement, then naturally degrades or is removed, preventing continuous uncontrolled genetic recombination while maintaining genome stability and eukaryote survival.
Solution Approach 2:
The patent extracts the restriction enzyme activity from the continuous gene expression system by introducing it as a transient protein. This allows precise temporal control where the enzyme is present only when needed for genome rearrangement, eliminating the problems of continuous expression while maintaining reliable DNA cleavage activity during the required window.
4Ease of manufacture
If back-crossing is used for genetic separation to remove introduced genes, then restriction enzyme expression can be eliminated, but the process is difficult or impossible in vegetatively propagating plants with poor seed-forming ability
Solution Approach 1:
The patent extracts the introduced gene requirement by directly introducing the restriction enzyme protein rather than the gene. This eliminates the need for genetic separation through back-crossing because no foreign gene remains in the genome to be segregated. The method is therefore universally applicable to all plants including vegetatively propagating species with poor seed-forming ability.
Solution Approach 2:
The patent inverts the conventional approach by introducing the functional protein product directly instead of the gene that would require removal. This reversal of the gene-to-protein pathway eliminates the need for subsequent genetic separation steps, making the method applicable to plants where traditional back-crossing is difficult or impossible.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach reduces the number of steps required to obtain eukaryotes with stable traits, avoids heat stress, and facilitates genetic diversity in plants with poor seed-forming abilities, allowing for efficient creation of diverse genome compositions without the need for gene introduction.
Implementation Method 1
introducing a protein having double-stranded DNA cleavage ability itself into the eukaryote or a part of the eukaryote, and rearranging the DNA of the eukaryote by the protein
Data Source
AI summary
A method includes introducing a protein having double-stranded DNA cleavage activity itself into the eukaryote or a part of the eukaryote, and rearranging DNA of the eukaryote by the protein in the eukaryote or a part of cells of the eukaryote.