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Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic
Bangzhu Chen1,2,3, Peng Gu1,2,3, Junshuang Jia1,4
1School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.
International Journal of Biological Sciences
|November 23, 2019
Summary
Researchers developed an efficient method to generate gene-edited Tibet minipigs for biomedical research. This technique overcomes low ovulation rates, enabling the creation of albinism and immunodeficiency models with high precision and no off-target mutations.
Area of Science:
- Genetics and Genomics
- Animal Models
- Biotechnology
Background:
- Tibet minipigs are valuable for research but have low ovulation rates, hindering genetic modification via zygote injection.
- Previous methods for generating genetically modified pigs are inefficient for Tibet minipigs due to reproductive challenges.
Purpose of the Study:
- To establish an efficient protocol for generating gene-edited Tibet minipigs.
- To create Tibet minipig models for albinism and immunodeficiency using CRISPR-Cas9 technology.
Main Methods:
- Utilized synchronized estrus instead of superovulation to collect zygotes.
- Optimized zygote preparation and co-injected clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs).
- Generated single (TYR) and dual (TYR-IL2RG, TYR-RAG1) gene knockouts.
Main Results:
- Successfully generated Tibet minipigs with TYR gene knockout (albinism) and TYR/IL2RG or TYR/RAG1 dual knockouts (albinism and immunodeficiency).
- Achieved high gene editing efficiencies: 75% for TYR, 25% for TYR-IL2RG, and 75% for TYR-RAG1.
- Confirmed germline transmission of targeted mutations and observed no detectable off-target mutations.
Conclusions:
- The developed method provides an efficient and precise approach for generating gene-edited Tibet minipigs.
- These gene-edited minipigs serve as valuable models for studying albinism, immunodeficiency, and other genetic conditions.
- This advancement facilitates future biomedical research utilizing Tibet minipigs.
