Generation and characterization of a conditional eNOS knock out mouse model for cell-specific reactivation of eNOS in gain-of-function studies

IF 3.2 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Anthea LoBue , Zhixin Li , Sophia K. Heuser , Junjie Li , Francesca Leo , Lukas Vornholz , Luke S. Dunaway , Tatsiana Suvorava , Brant E. Isakson , Miriam M. Cortese-Krott
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Abstract

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in the vessel wall regulates blood pressure and cardiovascular hemodynamics. In this study, we generated conditional eNOS knock out (KO) mice characterized by a duplicated/inverted exon 2 flanked with two pairs of loxP regions (eNOSinv/inv); a Cre-recombinase activity induces cell-specific reactivation of eNOS, as a result of a flipping of the inverted exon 2 (eNOSfl). This work aimed to test the efficiency of the Cre-mediated cell-specific recombination and the resulting eNOS expression/function. As proof of concept, we crossed eNOSinv/inv mice with DeleterCrepos (DelCrepos) mice, expressing Cre recombinase in all cells. We generated heterozygous eNOSfl/inv or homozygous eNOSfl/fl mice, and eNOSinv/inv littermate mice. We found that both eNOSfl/fl and eNOSfl/inv mice express eNOS and the overall expression level depends on the number of mutated alleles, while eNOSinv/inv mice did not show any eNOS expression. Vascular endothelial function was restored in eNOSfl/fl and eNOSfl/inv mice, as determined by ACh-dependent vasodilation of aortic rings. Cre-dependent reactivation of eNOS in eNOSfl/fl and eNOSfl/inv mice rescued eNOSinv/inv (phenotypically global eNOS KO) mice from hypertension. These findings demonstrate that eNOS expression is restored in eNOSfl/fl mice at comparable physiological levels of WT mice, and its functional activity is independent on the number of the reactivated alleles. Therefore, eNOSinv/inv mice are a useful model for studying the effects of conditional reactivation of eNOS and gene dosage effects in specific cells for gain-of-function studies.
生成条件性 eNOS 基因敲除小鼠模型并确定其特征,以便在功能增益研究中对 eNOS 进行细胞特异性再激活。
血管壁内皮一氧化氮合酶(eNOS)产生的一氧化氮(NO)调节血压和心血管血液动力学。在这项研究中,我们产生了条件性 eNOS 基因敲除(KO)小鼠,其特征是外显子 2 复制/倒置,侧翼有两对 loxP 区域(eNOSinv/inv);由于倒置的外显子 2 翻转(eNOSfl),Cre-重组酶活性诱导细胞特异性重新激活 eNOS。这项工作旨在测试 Cre 介导的细胞特异性重组的效率以及由此产生的 eNOS 表达/功能。作为概念验证,我们将 eNOSinv/inv 小鼠与 DeleterCrepos(DelCrepos)小鼠杂交,在所有细胞中表达 Cre 重组酶。我们产生了杂合子 eNOSfl/inv 或同合子 eNOSfl/fl 小鼠,以及 eNOSinv/inv 同窝小鼠。我们发现:(1)eNOSfl/fl 和 eNOSfl/inv 小鼠均表达 eNOS,总体表达水平取决于突变等位基因的数量,而 eNOSinv/inv 小鼠没有任何 eNOS 表达。(2)eNOSfl/fl 和 eNOSfl/inv 小鼠的血管内皮功能得到恢复,这是由主动脉环的 ACh 依赖性血管扩张决定的。(3)eNOSfl/fl 和 eNOSfl/inv 小鼠中 eNOS 的 Cre 依赖性再激活可挽救 eNOSinv/inv(表型上全局 eNOS KO)小鼠的高血压。这些研究结果表明,eNOSfl/fl 小鼠的 eNOS 表达恢复到了与 WT 小鼠相当的生理水平,而且其功能活性与重新激活的等位基因数量无关。因此,eNOSinv/inv 小鼠是研究 eNOS 条件性再激活效应和特定细胞中基因剂量效应的有用模型,可用于功能增益研究。
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来源期刊
Nitric oxide : biology and chemistry
Nitric oxide : biology and chemistry 生物-生化与分子生物学
CiteScore
7.50
自引率
7.70%
发文量
74
审稿时长
52 days
期刊介绍: Nitric Oxide includes original research, methodology papers and reviews relating to nitric oxide and other gasotransmitters such as hydrogen sulfide and carbon monoxide. Special emphasis is placed on the biological chemistry, physiology, pharmacology, enzymology and pathological significance of these molecules in human health and disease. The journal also accepts manuscripts relating to plant and microbial studies involving these molecules.
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