Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

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,). 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. The online version of this article (10.1186/s12967-018-1512-9) contains supplementary material, which is available to authorized users.

无毛小鼠已广泛应用于皮肤相关研究，而无毛猪由于其皮肤结构与人类相似，将成为皮肤相关研究及其他生物医学研究的理想模型。先前的研究表明，裸鼠的无毛表型是由磷脂酶C - δ1（PLCD1）表达不足引起的，PLCD1是Foxn1下游的一个关键分子，这促使我们培育PLCD1缺陷型猪。在本研究中，我们计划首先利用CRISPR/Cas9技术培育PLCD1基因敲除（KO）小鼠，这将为培育无毛的PLCD1基因敲除猪奠定坚实基础。 按照所述方法制备PLCD1的单向导RNA（sgRNAs）和Cas9信使RNA（mRNA）（邵，《自然实验手册》9:2493 - 2512）。通过将PLCD1 - sgRNA和Cas9 mRNA共显微注射到C57BL/6J受精卵的细胞质中，培育出PLCD1修饰的小鼠（F0代）。通过具有相似突变的F0代小鼠杂交获得纯合的PLCD1缺陷型小鼠（F1代）。 PLCD1修饰的小鼠（F0代）出生后出现渐进性脱毛，并且在PLCD1基因座第2外显子存在CRISPR/Cas9诱导的突变基因型，这表明sgRNA能有效引起导致毛发生长缺陷的突变。纯合的PLCD1缺陷型小鼠（F1代）腹部无毛，背部毛发稀疏。在PLCD1缺陷型小鼠中观察到毛囊数量减少、毛囊排列不规则且弯曲、表皮增生以及表皮分化紊乱等组织学异常。此外，PLCD1的表达水平显著降低，而参与毛囊分化的其他基因（如角蛋白1、角蛋白5、角蛋白13、兜甲蛋白和内披蛋白）在PLCD1缺陷型小鼠皮肤组织中的表达水平显著升高。 总之，我们利用CRISPR/Cas9技术获得了PLCD1基因敲除小鼠，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呈现完全无毛的表型。 本文的网络版（10.1186/s12967 - 2018 - 1512 - 9）包含补充材料，授权用户可获取。