Raymond Iona D, Pool Angela L, Vila Alejandro, Brecha Nicholas C
Department of Neurobiology, David Geffen UCLA School of Medicine, Los Angeles, CA 90095-1763, USA.
Vis Neurosci. 2009 Nov;26(5-6):453-65. doi: 10.1017/S095252380999023X. Epub 2009 Nov 23.
A DBA/2J (D2) transgenic mouse line with cyan fluorescent protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1-CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1-CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1-CFP transgene. CFP-fluorescent ganglion cells, 6-20 microm in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
为了在进行性青光眼过程中分析神经节细胞，构建了一种在神经节细胞中表达青色荧光蛋白（CFP）报告基因的DBA/2J（D2）转基因小鼠品系。Thy1-CFP D2（CFP-D2）品系是通过将发生色素性青光眼的D2品系与在神经节细胞中表达CFP的Thy1-CFP品系进行同基因杂交培育而成。对CFP-D2后代进行微卫星标记分析，证实了D2品系的isa和ipd位点的基因包含情况。这些位点内的特定突变会导致D2小鼠的黑素体蛋白功能失调和青光眼表型。聚合酶链反应分析证实了Thy1-CFP转基因的包含情况。直径为6-20微米的CFP荧光神经节细胞分布在所有视网膜区域，CFP突起贯穿内网状层，CFP荧光轴突位于纤维层和视神经乳头。使用针对神经节细胞标记物NF-L、NeuN、Brn3a和SMI32的抗体进行免疫组织化学，以确认CFP在神经节细胞中的表达。使用针对无长突细胞标记物HPC-1和ChAT的抗体进行免疫组织化学，以确认CFP在胆碱能无长突细胞中的弱表达。CFP-D2小鼠出现了青光眼表型，包括虹膜疾病、神经节细胞丢失、纤维层磨损和眼压升高。构建了一种具有表达CFP的神经节细胞的CFP-D2转基因品系，该品系具有（1）主要为D2遗传背景，（2）表达CFP的神经节细胞，以及（3）与年龄相关的进行性青光眼。该品系对于在青光眼进行性发展过程中体内和体外研究神经节细胞及其轴突的实验研究具有重要价值。
