Construction of a mouse model for alveolar type II epithelial cell-specific knockout of SENP1 gene based on the Cre-loxP recombinase system

doi:10.12307/2025.604

Abstract

Abstract: BACKGROUND: Previously, a SENP1 gene-silenced human alveolar epithelial cell line was successfully constructed in vitro, and the role of SENP1 in hyperoxic lung injury was investigated at the cellular level.
OBJECTIVE: To construct a mouse model of alveolar type II epithelial cell-specific knockout of SENP1 gene based on the Cre-loxP recombinase system.
METHODS: SENP1flox/- mice were self-crossed to obtain SENP1flox/flox and SENP1flox/- mice; Sftpc-Cre+/+ mice were crossed with wild-type mice to obtain more Sftpc-Cre+/- mice. Sftpc-Cre+/+ or offspring Sftpc-Cre+/- mice were crossed with SENP1flox/- or offspring SENP1flox/flox mice to obtain SENP1flox/-Sftpc-Cre+/- double heterozygous mice. SENP1flox/-Sftpc-Cre+/- mice were then crossed with SENP1flox/flox mice to obtain SENP1flox/floxSftpc-Cre+/- mice. The genomic DNA was extracted by tail clipping and amplified by PCR. The amplified product was subjected to agarose gel electrophoresis to determine the mouse genotypes. Lung tissues of SENP1flox/flox and SENP1flox/floxSftpc-Cre+/- mice were subjected to immunofluorescence double-labelling and western blot assay to verify the knockdown effect of SENP1 gene. Heart, liver, lung and kidney tissues of SENP1flox/flox and SENP1flox/floxSftpc-Cre+/- mice were stained with hematoxylin-eosin to observe the histomorphology of each organ in the two groups of mice.
RESULTS AND CONCLUSION: SENP1flox/floxSftpc-Cre+/- mice were correctly screened by agarose gel electrophoresis. Immunofluorescence double-labeling experiments showed that the mean fluorescence intensity of SENP1 was reduced in lung tissues of SENP1flox/floxSftpc-Cre+/- mice compared with that of SENP1flox/flox mice (P < 0.01) and no significant co-localization of SENP1 and Sftpc was observed (P < 0.01). Western blot results showed that SENP1 protein expression was reduced in lung tissues of SENP1flox/floxSftpc-Cre+/- mice compared with SENP1flox/flox mice (P < 0.001). Hematoxylin-eosin staining showed no significant alterations in the histomorphology of heart, liver, lung and kidney tissues in SENP1flox/flox and SENP1flox/floxSftpc-Cre+/- mice. This study successfully constructed alveolar type II epithelial cell-specific knockout SENP1 gene mice using the Cre-loxP recombinase system, which provides a good tool for the subsequent study of the role of SENP1 gene in lung diseases such as bronchopulmonary dysplasia and idiopathic pulmonary fibrosis, in which alveolar type II epithelial cells are the main damage cells.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: SENP1, Cre-loxP recombinase system, alveolar type II epithelial cells, conditional knockout, mouse

CLC Number:

Yang Kun, Zhang Rong, Wu Yue, Lei Xiaoping, Shen Yunchuan, Kang Lan, Dong Wenbin. Construction of a mouse model for alveolar type II epithelial cell-specific knockout of SENP1 gene based on the Cre-loxP recombinase system[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(14): 2943-2950.

Figures/Tables 6

2.1 小鼠SENP1基因型鉴定在SENP1基因5′arm反应体系中，SENP1纯合子(SENP1flox/flox)小鼠在398 bp处可见条带，杂合子(SENP1flox/-)小鼠在341 bp和398 bp处可见条带，野生型小鼠在341 bp处可见条带。在SENP1基因3′arm反应体系中，SENP1纯合子小鼠在438 bp处可见条带，杂合子小鼠在384 bp和438 bp处可见条带，野生型小鼠在384 bp处可见条带，见图2。 2.2 小鼠Sftpc-Cre基因型鉴定在Sftpc-Cre基因WT反应体系中，Sftpc-Cre纯合子小鼠(Sftpc-Cre+/+)在386 bp处无条带，Sftpc-Cre杂合子小鼠(Sftpc-Cre+/-)和野生型小鼠在386 bp处有条带；在Sftpc-Cre基因3′arm反应体系中，Sftpc-Cre+/+和Sftpc-Cre+/-小鼠在352 bp处有条带，野生型小鼠在352 bp处无条带，见图3。 2.3 小鼠SENP1flox/-Sftpc-Cre+/-双杂合基因型的鉴定图4为小鼠SENP1flox/-Sftpc-Cre+/-基因型鉴定结果。根据表4判读得出311，314，317，318为SENP1flox/-Sftpc-Cre+/-双杂合小鼠。 2.4 SENP1基因肺特异性敲除小鼠(SENP1flox/floxSftpc-Cre+/-)的鉴定图5为小鼠SENP1flox/floxSftpc-Cre+/-基因型鉴定结果。根据表4判读得出411，413和416为SENP1flox/flox Sftpc-Cre+/-小鼠。 2.5 SENP1基因肺特异性敲除效果免疫荧光双标实验显示，与SENP1flox/flox小鼠相比，SENP1flox/floxSftpc-Cre+/-小鼠肺组织中SENP1的平均荧光强度显著降低(P < 0.01)。见图6A，B。与SENP1flox/flox小鼠相比，SENP1flox/flox Sftpc-Cre+/-小鼠肺组织中SENP1和Sftpc未见明显的共定位(P < 0.01)。见图6A，C。Western blot结果显示，与SENP1flox/flox小鼠相比，SENP1flox/floxSftpc-Cre+/-小鼠肺组织中SENP1蛋白表达降低(P < 0.001)。见图7。以上结果说明SENP1基因在肺泡Ⅱ型上皮细胞中的敲除效果满意。 2.6 SENP1flox/flox小鼠及SENP1flox/floxSftpc-Cre+/-小鼠各脏器组织形态学改变肺组织苏木精-伊红染色显示SENP1flox/flox 小鼠和SENP1flox/floxSftpc-Cre+/-小鼠肺泡发育均良好，无炎症渗出，肺泡腔无异常增大，肺泡间隔无断裂，见图8A。与SENP1flox/flox小鼠相比，SENP1flox/floxSftpc-Cre+/-小鼠的平均线性截距和放射状肺泡计数均无统计学意义(P > 0.05)，见图8B，C。以上结果说明在生理情况下，肺泡Ⅱ型上皮细胞特异性敲除SENP1基因不会对肺发育造成影响。为了进一步观察肺泡Ⅱ型上皮细胞特异性敲除SENP1基因对小鼠其他脏器的影响，取SENP1flox/flox小鼠和SENP1flox/flox Sftpc-Cre+/-小鼠的心、肝和肾组织，通过苏木精-伊红染色观察到两组小鼠的心、肝和肾脏器发育良好，无明显病变，见图9。Western blot结果显示，SENP1flox/flox小鼠和SENP1flox/floxSftpc-Cre+/-小鼠的心、肝和肾组织中SENP1蛋白表达无统计学意义(P > 0.05)，见图10。这些结果说明在生理情况下，肺泡Ⅱ型上皮细胞特异性敲除SENP1基因不会对其他脏器造成影响。"

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