State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, China.
Sci Rep. 2018 Oct 18;8(1):15430. doi: 10.1038/s41598-018-32024-x.
The whey protein β-lactoglobulin (BLG) is a major milk allergen which is absent in human milk. Here, we for the first time generated DNA-free BLG bi-allelic knockout cow by zinc-finger nuclease (ZFNs) mRNA and produced BLG-free milk. According to the allergenicity evaluation of BLG-free milk, we found it can trigger lower allergic reaction of Balb/c mice including the rectal temperature drop and the allergen-specific immunoglobulin IgE production; BLG free-milk was easily digested by pepsin at 2 min, while BLG in control milk was still not completely digested after 60 min, and the binding of IgE from cow's milk allergy (CMA) patients to BLG free-milk was significantly lower than that to the control milk. Meanwhile, the genome sequencing revealed that our animal is free of off-target events. Importantly, editing animal genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to foods produced by genome edited animals. Finally, the ZFNs-mediated targeting in cow could be transmitted through the germline by breeding. These findings will open up unlimited possibilities of modifying milk composition to make it more suitable for human health and also improve the functional properties of milk.
乳清蛋白β-乳球蛋白（BLG）是一种主要的牛奶过敏原，在人乳中不存在。在这里，我们首次通过锌指核酸酶（ZFNs）mRNA 生成无 DNA 的 BLG 双等位基因敲除奶牛，并生产出无 BLG 的牛奶。根据对无 BLG 牛奶的变应原性评估，我们发现它可以引发 Balb/c 小鼠较低的过敏反应，包括直肠温度下降和过敏原特异性免疫球蛋白 IgE 的产生；胃蛋白酶在 2 分钟内即可轻易消化无 BLG 牛奶中的 BLG，而对照牛奶中的 BLG 在 60 分钟后仍未完全消化，并且来自牛乳过敏（CMA）患者的 IgE 与无 BLG 牛奶的结合明显低于与对照牛奶的结合。同时，基因组测序显示我们的动物没有脱靶事件。重要的是，在不将外源 DNA 引入细胞的情况下编辑动物基因组可能会减轻与经基因组编辑的动物生产的食品相关的监管问题。最后，通过繁殖可以将 ZFNs 介导的靶向在奶牛中传递到生殖系。这些发现将为改变牛奶成分以使其更适合人类健康开辟无限可能，同时也可以提高牛奶的功能特性。
