Inducible Acvr1 Mouse Model for Fibrodysplasia Ossificans Progressiva
Solution Overview
Problem
Current methods fail to establish a stable and viable mouse line with the perinatal lethal R206H mutation in the Acvr1 gene, which is associated with fibrodysplasia ossificans progressiva, due to embryonic or perinatal lethality, making it impossible to transmit this mutation through the germline.
Innovation Solution
A conditional genetic modification approach using site-specific recombinase recognition sites and an inducible recombinase, such as Cre-ERT2, to control the expression of the mutant Acvr1 allele, allowing for its induction only after birth and avoiding embryonic or perinatal expression, thereby creating a germline-transmissible allele that can model the disease phenotype.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the R206H mutation is introduced into the Acvr1 gene in the germline, then the mouse model can transmit the mutation through germline and produce progeny with FOP phenotype, but the mutation causes embryonic or perinatal lethality preventing stable line establishment
Solution Approach 1:
The Acvr1 gene is segmented into wild-type and mutant portions, with the mutant exon 5 (containing R206H) placed in antisense orientation. This segmentation allows the gene to be transmitted in germline in a non-lethal state while enabling controlled activation of the lethal mutant form in specific tissues or conditions.
Solution Approach 2:
The mutant exon is prepared in advance in an inactive state (antisense orientation) within the germline. This preliminary action allows the mutation to be inherited without immediate harmful effects, and the harmful effect is activated only when Cre recombinase is introduced to flip the exon into sense orientation.
Solution Approach 3:
Cre recombinase acts as an intermediary that controls the activation of the mutant exon. The recombinase mediates the conversion from inactive (antisense) to active (sense) orientation of the mutant exon, allowing precise control over when and where the lethal phenotype is expressed.
2Measurement precision
If the mutant Acvr1 allele is expressed during embryonic development, then the FOP phenotype can be studied in utero, but the expression causes perinatal lethality preventing viable offspring
Solution Approach 1:
The expression status of the mutant allele is made dynamic rather than static. The mutant exon can be switched from inactive to active state using Cre recombinase, allowing researchers to control when the phenotype is expressed. This enables studying the phenotype at specific time points without forcing continuous expression that would cause lethality.
Solution Approach 2:
The mutant allele expression is implemented as periodic rather than continuous. The Cre recombinase can be administered at specific time points to activate the mutant exon temporarily, allowing phenotype observation during specific windows while maintaining viability during non-expression periods.
3Productivity
If a stable mouse line with the R206H mutation is established, then continuous production of FOP phenotype progeny is possible, but the perinatal lethality prevents germline transmission
Solution Approach 1:
The harmful lethal effect is extracted from the germline transmission process. The mutant exon is kept in an inactive state during germline transmission, so only the genetic information is transmitted without the harmful phenotypic effect. The lethality is separated from the inheritance process itself.
Solution Approach 2:
The wild-type Acvr1 gene serves as a functional copy that compensates for the mutant allele during germline transmission. The mutant exon in antisense orientation acts as a dormant copy that can be activated later, allowing the mouse line to be maintained using the wild-type copy while the mutant copy is available for controlled activation.
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 enables the creation of a mouse model that can express the R206H mutation conditionally, allowing for the study of fibrodysplasia ossificans progressiva without the limitations of embryonic lethality, and facilitates the production of progeny with the desired phenotype, effectively mimicking the human disease.
Implementation Method 1
the nucleic acid construct comprises a mutant exon 5 (e5) encoding an R206H mutation, wherein the mutant e5 is present in antisense orientation and is flanked upstream and downstream by RSSR's oriented to direct an inversion of the mutant e5 to sense orientation; and the mouse comprises an inducible recombinase gene encoding a recombinase that is capable of inverting the antisense mutant e5 exon to sense orientation
Data Source
AI summary
A genetically modified mouse is provided that comprises a conditional Acvr1 allele that comprises a mutated exon that, upon induction, converts to a mutant exon phenotype, wherein the mutant exon phenotype includes ectopic bone formation. Mice comprising a mutant Acvr1 exon 5 in antisense orientation, flanked by site-specific recombinase recognition sites, are provided, wherein the mice further comprise a site-specific recombinase that recognizes the site-specific recombinase recognitions sites, wherein the recombinase is induced upon exposure of the mouse to tamoxifen. Upon exposure to tamoxifen, the recombinase is expressed and acts on the RRS-flanked mutant exon 5 and places the mutant exon 5 in sense orientation and deletes the wild-type exon.