CRISPR-Edited Cloned Pig Model for Humanized Biomedical Research
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Solution Overview
Problem
Current mouse models for immunodeficiency and liver injury are limited by genetic differences, size disparities with humans, short lifespan, low efficiency in simulating human physiological processes, and irreversible liver injury, making them unsuitable for advanced biomedical research, particularly in hepatocyte transplantation and tumor biology.
Innovation Solution
A dual pig model of severe immunodeficiency and liver injury is constructed using CRISPR/Cas9 gene editing technology to knock out RAG2, IL2Rγ, and FAH genes in porcine fetal fibroblasts, followed by somatic cell nuclear transfer, creating a RAG2−/−/IL2Rγ−/Y/FAH−/− triple-gene edited cloned pig, which exhibits immunodeficiency, liver injury, and phenotypic characteristics similar to humans.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If mouse models are used for immunodeficiency and liver injury research, then model construction is relatively simple, but genetic differences and size disparities limit their ability to simulate human physiological processes
Solution Approach 1:
The patent changes the species parameter from mouse to pig, selecting pigs whose physiological parameters (genome homology, body size, immune metabolism) are closer to humans, thereby improving simulation accuracy while maintaining model constructibility through established gene editing techniques
2Ease of manufacture
If traditional gene editing methods are used to construct dual mouse model, then model construction is achievable, but irreversible liver injury is caused and success rate is low
Solution Approach 1:
The patent applies preliminary action by knocking out the FAH gene in porcine fetal fibroblasts before somatic cell nuclear transfer, creating a liver injury model that is reversible through pharmacological intervention, thereby avoiding irreversible damage and improving construction success rate
Solution Approach 2:
The patent uses somatic cell nuclear transfer as an intermediary technique to introduce gene edits into the pig model without directly editing the animal in utero, allowing for better control and reversibility of the liver injury through pharmacological agents
3Ease of manufacture
If FAH−/−/Rag−/−/IL2Rγ−/− triple-gene simultaneously modified mouse model is constructed by hybridization mode, then model can be obtained, but construction period is long and production efficiency is low
Solution Approach 1:
The patent performs preliminary gene editing in cultured porcine fetal fibroblasts before nuclear transfer, allowing simultaneous knockout of multiple genes in a controlled in vitro environment, thereby dramatically reducing the time required compared to sequential hybridization breeding
Solution Approach 2:
The patent replaces the mechanical breeding process (hybridization and multiple generations of crossing) with a biotechnological approach (somatic cell nuclear transfer), substituting time-consuming biological reproduction with a more efficient cellular manipulation method
4Ease of manufacture
If mouse model is used, then model construction is relatively straightforward, but short lifespan limits the duration of research studies
Solution Approach 1:
The patent changes the lifespan parameter by selecting pigs, which have lifespans and developmental timelines much closer to humans than mice, thereby enabling long-term studies of liver disease progression, transplantation, and tumor biology that were previously limited by mouse lifespan
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
The dual pig model offers a more human-relevant research platform with improved efficiency, reversibility, and reduced inflammatory reactions, enabling advanced biomedical research applications in tumor biology, cell transplantation, and hepatocyte transplantation, with potential for batch production and market applications.
Implementation Method 1
Aiming at a coding region of a pig RAG2 gene, a 5th exon of an IL2Rγ gene, and a 2nd exon of an FAH gene, designing a corresponding sgRNA targeting vector and connecting same to a framework vector to obtain a pGL3-U6-sgRNA recombinant plasmid
Implementation Method 2
co-transfecting the pGL3-U6-sgRNA recombinant plasmid and a pST1374-NLS-flag-linker-Cas9 (Addgene no: 44758) into a porcine fetal fibroblast by a nuclear transfection instrument
Data Source
AI summary
The present disclosure relates to a sgRNA and constructing a dual pig model of severe immunodeficiency and liver injury and use thereof. The method comprises the steps of knocking out RAG2, IL2Rγ and FAH genes in a porcine fetal fibroblast by using a CRISPR/Cas9 technology, constructing a RAG2−/−/IL2Rγ−/Y/FAH−/− triple-gene edited cloned pig by using a somatic cell nuclear transfer technology, and obtaining a dual pig model of severe immunodeficiency and liver injury through phenotypic analysis and identification. The method overcomes the problems of long production period, low efficiency, irreversible damage, unsatisfactory use in a humanization degree and the like in the existing model construction technology, can realize a batch construction of the dual pig model of severe immunodeficiency and liver injury by a continuous cloning technology, and has great advantages and potential market application prospects in the related fields of tumor biology, cell transplantation, humanized animal models and the like.