Lcn8-Cre 敲入小鼠附睾初始段特异性 Cre 重组酶活性。
Epididymal initial segment-specific Cre recombinase activity in Lcn8-Cre knock-in mice.
机构信息
Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China.
Institute of Reproductive Medicine, Medical School of Nantong University, Nantong, China.
出版信息
Mol Biol Rep. 2021 Aug;48(8):6015-6023. doi: 10.1007/s11033-021-06604-6. Epub 2021 Jul 30.
BACKGROUND
Sperm acquire the ability to fertilize ova through a complex process of epididymal maturation. To identify the functions of genes expressed in the proximal epididymis, mouse models specific to this region are needed.
METHODS AND RESULTS
A Lcn8-Cre knock-in mouse line was generated using CRISPR/Cas9 technology. A 37 bp coding sequence of Lcn8 from the ATG start codon was replaced by an NLS-Cre-polyA cassette, resulting in Cre expression and the absence of Lcn8. Epididymal initial segment-specific Cre expression was identified using RT-PCR and western blotting, and the spatial-temporal Cre activity was further confirmed by using the Rosa26 reporter mice. Immunofluorescence staining showed that active Cre recombinase was present in the principal cells. Histological analyses of sperm and epididymides, and the four-month mating tests, were used to confirm that Cre expression did not affect normal development and male fecundity.
CONCLUSIONS
The novel Lcn8-Cre mice can be used to establish epididymal initial segment-specific conditional knock-out mouse models.
背景
精子通过复杂的附睾成熟过程获得受精卵子的能力。为了鉴定在附睾近端表达的基因的功能，需要针对该区域的特定小鼠模型。
方法和结果
使用 CRISPR/Cas9 技术生成了 Lcn8-Cre 敲入小鼠品系。从 ATG 起始密码子开始的 Lcn8 的 37bp 编码序列被 NLS-Cre-polyA 盒取代，导致 Cre 表达和 Lcn8 的缺失。使用 RT-PCR 和 Western blot 鉴定附睾起始段特异性 Cre 表达，并用 Rosa26 报告小鼠进一步确认时空 Cre 活性。免疫荧光染色显示，活性 Cre 重组酶存在于主细胞中。通过精子和附睾的组织学分析以及四个月的交配试验，证实 Cre 表达不会影响正常发育和雄性生育能力。
结论
新型 Lcn8-Cre 小鼠可用于建立附睾起始段特异性条件敲除小鼠模型。
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