Institute of Comparative Medicine & Laboratory Animal Center, Southern Medical University, Guangzhou, 510515, China.
Songshan Lake Pearl Laboratory Animal Sci. & Tech. Co., Ltd., Dongguan, 523808, China.
J Transl Med. 2018 May 25;16(1):141. doi: 10.1186/s12967-018-1512-9.
Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs.
Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493-2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation.
PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice.
In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don't display completely hairless phenotype as expected.
无毛小鼠已广泛应用于皮肤相关研究，而无毛猪将成为皮肤相关研究及其他生物医学研究的理想模型，因为其皮肤结构与人类相似。先前的研究表明，裸鼠的无毛表型是由于磷脂酶 C-δ 1（PLCD1）表达不足引起的，PLCD1 是 Foxn1 下游的一个重要分子，这促使我们生成 PLCD1 缺陷型猪。在本研究中，我们计划首先通过 CRISPR/Cas9 技术生成 PLCD1 敲除（KO）小鼠，为生成无毛 PLCD1 KO 猪奠定坚实基础。
sgRNA 和 Cas 9 mRNA 的生成如 Shao 等人在《自然—生物技术》（Nat Protoc 9:2493-2512, 2014）中所述进行。通过将 PLCD1-sgRNA 和 Cas9 mRNA 共微注射到 C57BL/6J 受精卵的细胞质中，生成 PLCD1 修饰小鼠（F0）。通过 F0 小鼠之间的相互交配，获得纯合 PLCD1 缺陷型小鼠（F1）。
PLCD1 修饰小鼠（F0）出生后出现进行性脱毛，PLCD1 基因座外显子 2 的 CRISPR/Cas9 诱导突变的基因型表明 sgRNA 可有效引起导致毛发生长缺陷的突变。纯合 PLCD1 缺陷型小鼠（F1）表现出腹部光秃，背部毛发稀疏。PLCD1 缺陷型小鼠的毛囊数量减少、排列不规则和弯曲的毛囊、表皮过度增生和表皮分化紊乱等组织学异常。此外，PLCD1 缺陷型小鼠皮肤组织中 PLCD1 的表达水平显著降低，而参与毛囊分化的其他基因（即 Krt1、Krt5、Krt13、角蛋白和 involucrin）的表达水平显著升高。
总之，我们通过 CRISPR/Cas9 技术实现了 PLCD1 KO 小鼠，为毛发发育研究提供了一种新的动物模型，尽管纯合子并未如预期那样表现出完全无毛表型。
Zotero 插件
