Differentiation-Dependent Self-Deleting Cassette for Selectable Marker Excision
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Solution Overview
Problem
Current methods for removing selectable markers from genetically modified cells and animals are inefficient, requiring multiple rounds of breeding and screening to achieve complete excision, which can be time-consuming and introduce unwanted genetic backgrounds, complicating the interpretation of knockout phenotypes.
Innovation Solution
A differentiation-dependent expression system using promoters like Gata6 or Blimp1, and miRNA recognition sequences to control the expression of site-specific recombinases, ensuring the selective removal of selection cassettes only upon cell differentiation, thereby facilitating efficient and complete excision in all tissues without the need for extensive breeding or screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional breeding and screening methods are used to remove selectable markers, then complete excision can be achieved, but the process is time-consuming and requires multiple rounds of breeding
Solution Approach 1:
The patent incorporates a recombinase gene and recognition sites into the targeting vector before introducing it into ES cells. This preliminary setup enables automatic excision of the selection cassette during subsequent breeding, eliminating the need for multiple manual screening rounds and significantly reducing the time required to achieve complete excision.
Solution Approach 2:
The selection cassette is designed with flanking recognition sites that enable self-excision through recombinase-mediated recombination. The system automatically removes the selection cassette during the breeding process without requiring external intervention or manual screening, thus saving time while ensuring complete excision.
2Reliability
If traditional breeding methods are used to remove selectable markers, then excision can be achieved, but unwanted genetic backgrounds are introduced
Solution Approach 1:
The patent extracts the selection cassette from the final knockout allele by incorporating flanking recognition sites that enable its removal through recombinase-mediated recombination. This extraction eliminates the harmful presence of the selection cassette and prevents the introduction of unwanted genetic backgrounds during breeding, as the cassette is automatically removed in a single step.
Solution Approach 2:
The recombinase enzyme acts as an intermediary that facilitates the precise excision of the selection cassette without requiring multiple rounds of breeding. This single-step recombination process eliminates the need for extensive breeding that would otherwise introduce unwanted genetic backgrounds, thereby maintaining genetic purity.
3Reliability
If selection cassettes are retained in knockout alleles, then drug selection can be maintained, but expression of neighboring genes may be interfered with
Solution Approach 1:
The patent incorporates flanking recognition sites into the selection cassette design before introducing it into ES cells. This preliminary configuration enables the cassette to be automatically excised after drug selection is complete, preventing any potential interference with neighboring gene expression while maintaining the necessary drug selection capability during the selection process.
Solution Approach 2:
The selection cassette is temporarily retained during the drug selection process to ensure reliable selection of correctly targeted ES cells. After selection is complete, the cassette is automatically excised through recombinase-mediated recombination, discarding the potentially harmful element while having already served its useful function. This ensures both reliable drug selection and prevention of subsequent interference with neighboring genes.
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 allows for the reliable and efficient excision of selection cassettes in genetically modified cells and animals, reducing the complexity of breeding and maintaining genetic purity, thus enhancing the accuracy of knockout mouse models.
Implementation Method 1
the 3'-UTR comprises an miRNA recognition site, wherein the miRNA recognition site is recognized by an miRNA that is active in an undifferentiated cell but is not active in a differentiated cell
Data Source
AI summary
Targeting constructs and methods of using them are provided for differentiation-dependent modification of nucleic acid sequences in cells and in non-human animals. Targeting constructs comprising a promoter operably linked to a recombinase are provided, wherein the promoter drives transcription of the recombinase in an differentiated cell but not an undifferentiated cell. Promoters include Blimp1, Prm1, Gata6, Gata4, Igf2, Lhx2, Lhx5, and Pax3. Targeting constructs with a cassette flanked on both sides by recombinase sites can be removed using a recombinase gene operably linked to a 3′-UTR that comprises a recognition site for an miRNA that is transcribed in undifferentiated cells but not in differentiated cells. The constructs may be included in targeting vectors, and can be used to automatically modify or excise a selection cassette from an ES cell, a non-human embryo, or a non-human animal.