Genetically Engineered Goats as Efficient Mammary Gland Bioreactors for Production of Recombinant Human Neutrophil Peptide 1 Using CRISPR/Cas9

文献类型： 外文期刊

第一作者： Li, Dongxu

作者： Li, Dongxu;Wang, Jingang;Wang, Feng;Wan, Yongjie;Guo, Rihong;Chen, Fang;Guo, Rihong;Chen, Fang

作者机构： Nanjing Agr Univ, Coll Anim Sci & Technol, Jiangsu Livestock Embryo Engn Lab, Nanjing 210095, Peoples R China;Jiangsu Prov Engn Res Ctr Precis Anim Breeding, Nanjing 210014, Peoples R China;Jiangsu Acad Agr Sci, Inst Anim Sci, Nanjing 210014, Peoples R China

关键词： CRISPR/Cas9; goat; HNP1; beta-casein; antibacterial activity

期刊名称：BIOLOGY-BASEL （ 2023影响因子：3.6； 五年影响因子：3.8 ）

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年卷期： 2024 年 13 卷 6 期

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收录情况： SCI

摘要： Simple Summary Mammary bioreactors represent a promising method for the production of recombinant proteins. In this study, the human neutrophil peptide 1 (HNP1) sequence was knocked into the seventh exon of the goat beta-casein (CSN2) gene under the control of the CSN2 promoter using CRISPR/Cas9 technology. A mixture of Cas9 mRNA, sgRNA, and a homologous plasmid including the T2A-HNP1 sequences were microinjected into the embryos of donor goat and then transplanted into recipient goats through embryo transfer technology. This allowed the HNP1 gene to be expressed in the offspring, facilitating the production of antimicrobial peptide proteins through the goat's mammary glands. This experiment successfully produced genetically edited goats that secrete HNP1 with mammary-specific characteristics, providing a scientific basis for the further promotion of gene-editing technology and the development of new transgenic goat breeds with antimicrobial components in their milk.Abstract Mammary gland bioreactors are promising methods for recombinant protein production. Human neutrophil peptide 1 (HNP1) exhibits antibacterial and immune-modulating properties. This study aims to establish a method to generate goats secreting HNP1 using the mammary gland as bioreactors. HNP1 transgenic goats were generated by using CRISPR/Cas9 technology to knock-in (KI) the HNP1 sequence into exon 7 of the goat beta-casein (CSN2) gene under the control of the CSN2 promoter. One-cell stage embryos were cytoplasmically injected with a mixture of Cas9 mRNA, sgRNA, and a homologous plasmid including the T2A-HNP1 sequences, followed by transfer to recipient goats. A total of 22 live offspring goats were delivered, and 21 of these goats (95.45%) exhibited targeted edits at the CSN2 locus, and 2 female goats (9.09%) demonstrated successful HNP1 integration. Western blot and ELISA analyses confirmed the presence of HNP1 protein at high levels in the milk of these HNP1-positive goats, with mean concentrations of 22.10 mu g/mL and 0.0092 mu g/mL during the initial 60 days of lactation. Furthermore, milk from these transgenic goats exhibited notable antibacterial activity against Escherichia coli and Staphylococcus aureus, demonstrating the functionality of the expressed HNP1 protein. In conclusion, we established an efficient method for developing new transgenic goat lines as a mammary gland bioreactor, and the bioactive HNP1 protein secreted by the transgenic goat has the potential to combat microbial resistance.

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