TALEN-Mediated RAG2 Gene Targeting in Miniature Pigs
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Solution Overview
Problem
Current methods for producing animal models for severe combined immunodeficiency (SCID) are limited due to the lack of effective human-like models, hindering research in cancer, cell transplantation, and drug development.
Innovation Solution
A method using TALEN-mediated gene targeting to introduce biallelic mutations in the RAG2 gene of miniature pigs, achieved through somatic cell nuclear transplantation, creating SCID-like pigs with specific immunological deficiencies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional gene targeting methods are used to produce animal models for SCID, then the models lack human-like physiological properties, but developing effective human-like models is hindered by limited availability
Solution Approach 1:
The patent changes the species parameter from conventional rodent models to miniature pigs, which have physiological properties much more similar to humans. This parameter change resolves the contradiction by providing human-like physiological similarity while maintaining the feasibility of genetic modification through established porcine genetic engineering techniques
Solution Approach 2:
The patent replaces conventional gene targeting methods with TALEN-mediated gene editing, which provides more precise and efficient gene modification. This substitution enables reliable production of SCID pig models with specific RAG2 gene mutations, resolving the contradiction between model reliability and production ease
2Reliability
If TALEN-mediated gene targeting is used to introduce biallelic mutations in RAG2 gene, then SCID pigs with high reliability are produced, but the gene editing process complexity increases
Solution Approach 1:
The patent uses TALEN proteins as intermediary molecules that specifically recognize and bind to the RAG2 gene target sequence. This intermediary approach enables precise biallelic mutation induction without requiring complex CRISPR-Cas9 systems, resolving the contradiction by achieving high reliability through a relatively simple protein-based gene editing mechanism
Solution Approach 2:
The patent divides the gene editing process into discrete steps: TALEN construction with specific recognition sequences, transfection into porcine cells, selection of successfully edited cells, and somatic cell nuclear transfer. This segmentation makes the complex gene editing process manageable and reproducible, resolving the contradiction between reliability and complexity
3Reliability
If somatic cell nuclear transplantation is used to produce mutant embryos, then SCID pigs are successfully generated, but the overall production process time and complexity increase
Solution Approach 1:
The patent performs gene editing in somatic cells before nuclear transfer, allowing selection and verification of successfully edited cells in advance. This preliminary action ensures that only cells with the desired RAG2 mutations are used for SCNT, guaranteeing SCID pig generation success while optimizing the overall production timeline by avoiding rejections later in the process
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 approach effectively produces SCID pigs with targeted RAG2 mutations, mimicking human SCID, facilitating research in immunodeficiency, cancer, and drug development by providing a reliable and immunocompromised model.
Implementation Method 1
inducing a biallelic mutation by targeting a transcription activator-like effector nuclease (TALEN) to a TALEN-recognizing sequence region
Implementation Method 2
producing the mutant embryos by using the cells with the induced biallelic mutation for somatic cell nuclear transplantation
Implementation Method 3
a reporter vector including a monomeric red fluorescent protein (RFP) gene, a targeting sequence of the programmable nuclease, an enhancer green fluorescent protein (GFP) gene
Data Source
Figure 1A~2A
Figure 2B~2D
Figure 2E~2F
AI summary
There are provided to a Rag-2 (Recombination activating gene 2) gene targeting vector, a method for producing SCID-like miniature pigs introduced with the vector, and a use thereof.