Napoli Francesca R, Li Xiaodong, Hurtado Alan A, Levine Edward M
Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN, 37232, USA.
Program in Chemical and Physical Biology, Vanderbilt University, Nashville, TN, 37232, USA.
Eye Brain. 2024 Nov 23;16:115-131. doi: 10.2147/EB.S480996. eCollection 2024.
Visual System Homeobox 2 () is a transcription factor expressed in the developing retina that regulates tissue identity, growth, and fate determination. Several mutations in the gene exist in mice, including a spontaneous nonsense mutation and two targeted missense mutations originally identified in humans. Here, we expand the genetic repertoire to include a reporter allele ( ) designed to express beta-Galactosidase (bGal) and simultaneously disrupt function (knock-in/knock-out).
We generated a reporter allele with an in-frame fusion to the coding sequence immediately following exon 2. Germline transmission was assessed with genomic DNA PCR and Western blot analysis was used to describe VSX2 expression from the mutant allele (). Eye size quantification and immunohistology were used to describe the embryonic and postnatal retinal phenotypes of homozygous and heterozygous mice. The contribution of to mutant microphthalmia was probed with the semi-dominant negative allele.
The retinal expression pattern of bGal is concordant with VSX2, and the mutant allele is recessive. homozygous mice have congenital bilateral microphthalmia accompanied by defects in retinal development including ectopic expression of non-retinal genes, reduced proliferation, delayed neurogenesis, aberrant tissue morphology, and an absence of bipolar interneurons - all hallmarks of loss-of-function. The allele reduced the severity of microphthalmia caused by the allele. Unexpectedly, the mutant VSX2 protein is stably expressed, and there are subtle differences in eye size and early retinal neurogenesis when compared to the null mutant, .
The perdurance of the mutant VSX2 protein combined with subtle deviations from the null phenotype leaves open the possibility that allele is not a complete knock-out. The allele exhibits loss-of-function characteristics and adds to the genetic toolkit for understanding function.
视觉系统同源盒2（VSX2）是一种在发育中的视网膜中表达的转录因子，可调节组织特性、生长和命运决定。VSX2基因在小鼠中存在多种突变，包括一个自发的无义突变和最初在人类中鉴定出的两个靶向错义突变。在此，我们扩展了该基因库，纳入了一个VSX2报告等位基因（VSX2LacZ），其设计用于表达β-半乳糖苷酶（bGal）并同时破坏VSX2功能（敲入/敲除）。
我们生成了一个VSX2报告等位基因，其与外显子2之后的VSX2编码序列进行了读码框内融合。通过基因组DNA聚合酶链反应（PCR）评估种系传递情况，并使用蛋白质免疫印迹分析来描述来自突变等位基因（VSX2LacZ）的VSX2表达。通过眼睛大小量化和免疫组织学来描述VSX2纯合和杂合小鼠的胚胎期和出生后视网膜表型。使用半显性负性VSX1等位基因探究VSX1对VSX2突变性小眼症的影响。
bGal的视网膜表达模式与VSX2一致，且突变等位基因是隐性的。VSX2纯合小鼠患有先天性双侧小眼症，并伴有视网膜发育缺陷，包括非视网膜基因的异位表达、增殖减少、神经发生延迟、异常的组织形态以及双极中间神经元缺失——所有这些都是VSX2功能丧失的特征。VSX1等位基因降低了由VSX2等位基因引起的小眼症的严重程度。出乎意料的是，突变的VSX2蛋白稳定表达，与无效突变体VSX2-/-相比，眼睛大小和早期视网膜神经发生存在细微差异。
突变的VSX2蛋白的持久性以及与无效表型的细微偏差使得VSX2LacZ等位基因可能不是完全敲除的可能性仍然存在。VSX2LacZ等位基因表现出功能丧失的特征，并为理解VSX2功能增添了遗传工具。
