Two-Step LoxP Introduction for Conditional Knockout Animals
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Solution Overview
Problem
Current methods for producing conditional knockout animals using the Cre/loxP system have low floxing efficiencies due to preferential chromosome deletion when two loxP sequences are introduced simultaneously.
Innovation Solution
Introducing the first and second recombinase recognition sequences at different timings in two separate steps into the chromosome of a non-human animal cell, such as using CRISPR/Cas for introducing loxP sequences, to achieve higher floxing efficiencies and reduce chromosomal deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If two loxP sequences are introduced simultaneously into a chromosome of a fertilized egg, then the preparation time for floxed mice is shortened, but the floxing efficiency becomes low due to preferential chromosome deletion
Solution Approach 1:
The patent divides the simultaneous introduction of two loxP sequences into two separate sequential steps. First, one loxP sequence is introduced into the chromosome, and then the second loxP sequence is introduced in a subsequent step. This segmentation prevents preferential chromosome deletion that occurs when both sequences are introduced simultaneously, thereby maintaining high floxing efficiency while still achieving relatively quick preparation compared to traditional ES cell methods.
2Device complexity
If two loxP sequences are introduced at the same time, then the process is simplified, but chromosomal deletions occur preferentially reducing successful floxed offspring
Solution Approach 1:
The patent introduces the first loxP sequence into the chromosome as a preliminary step before introducing the second loxP sequence. This preliminary action establishes a stable intermediate state where one loxP marker is already present, which prevents preferential chromosome deletion when the second sequence is subsequently introduced. The sequential approach maintains process simplicity while eliminating the harmful chromosomal deletions associated with simultaneous introduction.
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 significantly enhances the efficiency of producing floxed animals by increasing the number of successful floxed offspring and reducing chromosomal deletions, as demonstrated by the two-step electroporation method which achieves a 4.6 times higher floxing efficiency compared to conventional one-step methods.
Implementation Method 1
using CRISPR/Cas for introducing loxP sequences
Implementation Method 2
as demonstrated by the two-step electroporation method
Data Source
AI summary
A method of producing a conditional knockout animal, and techniques related thereto, e.g., a method of efficiently producing a floxed animal, are provided. By introducing recombinase recognition sequences such as loxP into both ends of a target region on a chromosome at different timings, an animal having the pair of recombinase recognition sequences on the chromosome, such as a floxed animal, is produced.