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| CRISPR/Cas9系统介导的人乳铁蛋白基因在山羊β-乳球蛋白基因座定点敲入 |
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宋绍征1,张婷2,潘生强1,陆睿3,成勇2*,周鸣鸣1*
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1.无锡太湖学院 护理学院, 江苏 无锡 214000;2.扬州大学 兽医学院/江苏省转基因动物制药工程研究中心, 江苏 扬州 225009;3.江苏食品药品职业技术学院 制药工程学院, 江苏 淮安 223003
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| 摘要: |
| 为研究山羊乳蛋白基因编辑，实现羊乳“人源化”改造。利用CRISPR/Cas9系统敲除山羊乳中致敏原β-乳球蛋白(BLG)基因，同时在BLG基因座定点敲入人乳铁蛋白(hLF)基因。针对山羊BLG基因第一外显子设计构建sgBLG/Cas9表达载体经电转染山羊胎儿成纤维细胞验证其编辑活性；将打靶载体BLC14与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞，经筛选检测获得 BLG-/hLF+打靶细胞株作为供核细胞用于山羊体细胞核移植；通过剖宫产获取克隆胎儿并验证其中靶情况。结果表明:sgBLG/Cas9编辑山羊BLG位点致突变率约为30%~35%；共筛选72 株药物抗性细胞株，其中有37 株为基因打靶细胞，打靶效率为51.4%(37/72)；挑取形态较好的7株打靶细胞用于核移植，共制备416 枚重构胚，移植30 只受体山羊；30-35 日龄 B超诊断有13 只受孕，妊娠率为43.3%(13/30)；剖宫产获取3 只35 日龄克隆胎儿均验证为BLG-/hLF+基因型，并建立了BLG-/hLF+靶向性修饰细胞系。因此，利用CRISPR/Cas9系统可以获得BLG基因座定点打靶hLF基因的转基因克隆山羊，该研究为培育羊乳中低致敏原和富含hLF功能营养成分的转基因山羊新品系提供了科学依据。 |
| 关键词: CRISPR/Cas9 β-乳球蛋白 人乳铁蛋白 基因打靶 核移植 |
| DOI：10.11841/j.issn.1007-4333.2020.07.11 |
| 分类号: |
| 基金项目:国家转基因生物新品种培育重大专项(2014ZX08008-004)；江苏省高校自然科学基金面上项目(19KJB180030)；无锡市科协软科学研究重点课题(KX-19-B28) |
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| hLF gene knock-in at the BLG locus of goat by CRISPR/Cas9 system |
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SONG Shaozheng1,ZHANG Ting2,PAN Shengqiang1,LU Rui3,CHENG Yong2*,ZHOU Mingming1*
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1.School of Nursing, Wuxi Taihu University, Wuxi 214000, China;2.College of Veterinary Medicine/Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming, Yangzhou University, Yangzhou 225009, China;3.School of Pharmaceutical Engineering, Jiangsu Food and Pharmaceutical Science College, Huaian 223003, China
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| Abstract: |
| In order to study the gene editing of goat milk protein and realize the “humanization” transformation of goat milk, the β-lactoglobulin(BLG)gene was knocked out and the human lactoferrin(hLF)gene was knocked in at BLG locus in goat by CRISPR/cas9 system. A sgBLG/Cas9 expression vector for goat BLG exon Ⅰrecognition site was designed and constructed, which was electrotransfected into the goat fetal fibroblast cells to verify the editing activity. The vector BLC14 and sgBLG/Cas9 were co-transfected into goat fetal fibroblasts, and the BLG-/hLF+ targeting cells were chosen as donor cells for goat somatic cell nuclear transfer. The cloned fetus was obtained by cesarean section and the target condition was verified. The results showed that: The mutagenicity of sgBLG/Cas9 editing goat BLG locus was 25%-30%; A totoal of 72 G418-resistant cells were screened, 37 of which were gene targeting cells, and the targeting efficiency was 51. 4%(37/72); In this study, seven gene target cells having better morphology appearances were selected for nuclear transplantation, and 416 reconstructed embryos were transferred into 30 recipient goats. There were 13 pregnancies confirmed by B-ultrasound in 30-35 days, and pregnancy rate was 43. 3%(13/30). All three 35-day-old cloned fetuses by cesarean section, and they were all identified as BLG-/hLF+ genotype and the targeted modified cell lines were established. In conclusion, the hLF gene knock-in at BLG locus of goat could be obtained by CRISPR/cas9 system, which provided a scientific basis for cultivating transgenic goat lines of low allergen and rich hLF in the milk, and also laid a foundation for milk protein gene editing and molecular genetic breeding. |
| Key words: CRISPR/cas9 β-lactoglobulin human lactoferrin gene targeting nuclear transplantation |
