应用Cre-loxP系统构建表皮细胞中p75神经营养因子受体基因条件性敲除小鼠模型及其鉴定

Construction and identification of mouse model with conditional knockout of p75 neurotrophin receptor gene in epidermal cells by Cre-loxP system

目的应用Cre-loxP系统构建表皮细胞中p75神经营养因子受体(p75NTR )基因条件性敲除的小鼠模型并进行鉴定。方法应用Cre-loxP系统将5只6~8周龄雌雄不限(用于繁殖的小鼠鼠龄和性别下同)p75NTRflox/flox转基因C57BL/6J小鼠和5只角蛋白14启动子驱动(KRT14-)Cre+/-转基因C57BL/6J小鼠进行饲养和杂交。从繁育产生的第1代小鼠中筛选出5只p75NTRflox/+·KRT14-Cre+/-小鼠与5只p75NTRflox/flox小鼠进行交配,获得第2代小鼠。第2代小鼠出生20~25d,切取每只小鼠鼠尾,通过PCR法进行基因型鉴定。分别选取经鉴定后生长至6周龄的p75NTR基因完全条件性敲除小鼠、野生型小鼠各4只处死,切取腹部皮肤组织和脑组织,经免疫组织化学染色对比观察p75NTR在2种小鼠2种组织中的表达情况;另切取腹部皮肤组织,经苏木精-伊红染色观察2种小鼠组织形态学变化。结果(1)共繁育第2代小鼠20只,其中4只小鼠基因型为p75NTRflox/flox·KRT14-Cre+/-(p75NTR-/-),即p75NTR基因完全条件性敲除小鼠;5只小鼠基因型为p75NTRflox/+·KRT14-Cre+/-,即p75NTR基因部分条件性敲除小鼠;5只小鼠基因型为p75NTRflox/flox·KRT14-Cre-/-,6只小鼠基因型为p75NTRflox/+·KRT14-Cre-/-,均为野生型小鼠。(2)p75NTR基因完全条件性敲除小鼠皮肤表皮组织中未见p75NTR表达,野生型小鼠皮肤表皮组织中可见较多p75NTR阳性表达;p75NTR基因完全条件性敲除小鼠和野生型小鼠脑组织中均有丰富p75NTR阳性表达。(3)p75NTR基因完全条件性敲除小鼠和野生型小鼠皮肤表皮组织均无生长异常情况且毛囊结构均完整。结论应用Cre-loxP系统能够成功构建表皮细胞中p75NTR基因条件性敲除小鼠模型且小鼠皮肤组织形态无明显变化。

Objective To construct and identify a mouse model with conditional knockout (cKO) of p75 neurotrophin receptor (p75NTR-cKO) gene in epidermis cells by Cre-loxP system. Methods Five p75NTRflox/flox transgenic C57BL/6J mice (aged 6-8 weeks, male and female unlimited, the age and sex of mice used for reproduction were the same below) and five keratin 14 promotor-driven (KRT14-) Cre+/- transgenic C57BL/6J mice were bred and hybridized via Cre-loxP system. Five p75NTRflox/+·KRT14-Cre+/- mice selected from the first generation of mice were mated with five p75NTRflox/flox mice to obtain the second generation hybrids. After the second generation mice were born 20-25 days, the parts of the mice tail were cut off to identify the genotype by polymerase chain reaction method. Four p75NTR gene complete cKO mice (6 weeks old) and 4 wild-type mice (6 weeks old) were selected and sacrificed respectively. The abdominal skin tissue and brain tissue were excised to observe the expression of p75NTR in the two tissue of two types of mice by immunohistochemical staining. The abdominal skin tissue of two types of mice was obtained to observe the histomorphological changes by hematoxylin and eosin staining. Results (1) Twenty second generation mice were bred. The genotype of 4 mice was p75NTRflox/flox·KRT14-Cre+/-(p75NTR-/-), i. e. p75NTR gene complete cKO mice;the genotype of 5 mice was p75NTRflox/+·KRT14-Cre+/-, i. e. p75NTR gene partial cKO mice;the genotype of 5 mice was p75NTRflox/flox·KRT14-Cre-/-, and that of 6 mice was p75NTRflox/+·KRT14-Cre-/-, all of which were wild-type mice.(2) The expression of p75NTR was negative in skin epidermis tissue of p75NTR gene complete cKO mice, while numerous p75NTR positive expression was observed in skin epidermis tissue of wild-type mice. Abundant p75NTR positive expression was observed in brain tissue of both wild-type mice and p75NTR gene complete cKO mice.(3) There was no abnormal growth of skin epidermis tissue in both wild-type mice and p75NTR gene complete cKO mice, with intact hair follicle structure. Conclusions Applying Cre-loxP system can successfully construct a p75NTR-cKO mice model in epidermis cells without obvious changes in skin histomorphology.