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Characterization of the two inducible Cre recombinase-based mouse models NG2- CreERTM and PDGFRb-P2A-CreERT2 for pericyte labeling in the retina
Current Eye Research ( IF 2 ) Pub Date : 2021-11-10 , DOI: 10.1080/02713683.2021.2002910
Daniela Mayr 1 , Julia Preishuber-Pflügl 1 , Andreas Koller 1 , Susanne M Brunner 1 , Christian Runge 1 , Anja-Maria Ladek 1 , Francisco J Rivera 2, 3, 4 , Herbert A Reitsamer 1, 5 , Andrea Trost 1
Affiliation  

Purpose/aim of the study

Pericytes (PCs), located abluminal of endothelial cells on capillaries, are essential for vascular development and stability. They display a heterogeneous morphology depending on organ localization, differentiation state and function. Consequently, PCs show a diverse gene expression profile, impeding the usage of a unique PC marker and therefore the distinct identification of PCs. Inducible reporter mouse models represent an important tool for investigating the fate of PCs under physiological and pathophysiological conditions. PC-specific expression efficiency of the fluorescence reporter tdTomato following tamoxifen induction was analyzed and compared in two inducible Cre recombinase-expressing mouse models under control of the NG2 and PDGFRb promotor.

Material and methods

The NG2-CreERTM-tdTomato and the PDGFRb-P2A-CreERT2-tdTomato mice were treated with tamoxifen at three defining time points of retinal vascular development: postnatal days (P)5, P10/11/12 and P48/49/50/51. TdTomato reporter induction efficiency was determined by analyzing retinal whole mounts utilizing confocal microscopy, using the antibodies Anti-neural/glial antigen 2 (PCs), Anti-Collagen IV (basement membrane) and Anti-Glutamine Synthetase (Müller glial cells).

Results

Tamoxifen induction at the three different time points resulted in PC-specific expression of tdTomato in both reporter models. In the NG2-CreERTM-tdTomato mouse, the induction efficiency ranged from 21.9 to 35.5%. In the PDGFRb-P2A-CreERT2-tdTomato mouse an induction efficiency between 78.9 and 94.1% was achieved. TdTomato expression in the retina was restricted to PCs and vascular smooth muscle cells in the NG2-CreERTM-tdTomato mouse, however, in the PDGFRb-P2A-CreERT2-tdTomato mouse, tdTomato was also expressed in Müller glial cells.

Conclusion

Both reporter mouse models represent promising tools for fate mapping studies of PCs. While the NG2-CreERTM-tdTomato mouse reveals very specific labeling of PCs in the retina, its induction efficiency is lower compared to the PDGFRb-P2A-CreERT2-tdTomato mouse. Although the latter revealed a high percentage of tdTomato-positive PCs in the retina, additional labeling of Müller cells potentially hampers analysis of reporter-positive PCs.



中文翻译:

用于视网膜周细胞标记的两种基于 Cre 重组酶的诱导型小鼠模型 NG2-CreERTM 和 PDGFRb-P2A-CreERT2 的表征

研究目的/目的

周细胞 (PC) 位于毛细血管内皮细胞的腔外,对血管发育和稳定性至关重要。它们根据器官定位、分化状态和功能表现出异质形态。因此,PC 显示出多样化的基因表达谱,阻碍了独特 PC 标记的使用,因此阻碍了 PC 的独特识别。诱导型报告小鼠模型是研究生理和病理生理条件下 PC 命运的重要工具。在 NG2 和 PDGFRb 启动子控制下的两种可诱导 Cre 重组酶表达小鼠模型中分析和比较了他莫昔芬诱导后荧光报告基因 tdTomato 的 PC 特异性表达效率。

材料与方法

NG2-CreER TM -tdTomato 和 PDGFRb-P2A-CreER T2 -tdTomato 小鼠在视网膜血管发育的三个定义时间点用他莫昔芬治疗:出生后天 (P)5、P10/11/12 和 P48/49/50 /51。TdTomato 报告基因的诱导效率是通过使用共聚焦显微镜分析视网膜整个支架,使用抗体抗神经/神经胶质抗原 2 (PC)、抗胶原蛋白 IV(基底膜)和抗谷氨酰胺合成酶(Müller 神经胶质细胞)来确定的。

结果

他莫昔芬在三个不同时间点的诱导导致两种报告模型中 tdTomato 的 PC 特异性表达。在 NG2-CreER TM -tdTomato 小鼠中,诱导效率为 21.9% 至 35.5%。在 PDGFRb-P2A-CreER T2 -tdTomato 小鼠中,诱导效率在 78.9% 和 94.1% 之间。视网膜中的 TdTomato 表达仅限于 NG2-CreER TM -tdTomato 小鼠中的 PC 和血管平滑肌细胞,然而,在 PDGFRb-P2A-CreER T2 -tdTomato 小鼠中,tdTomato 也在 Müller 神经胶质细胞中表达。

结论

两种报告鼠标模型都代表了 PC 命运映射研究的有前途的工具。虽然 NG2-CreER TM -tdTomato 小鼠在视网膜中显示出非常特异性的 PC 标记,但与 PDGFRb-P2A-CreER T2 -tdTomato 小鼠相比,其诱导效率较低。尽管后者显示视网膜中 tdTomato 阳性 PC 的百分比很高,但对 Müller 细胞的额外标记可能会阻碍对报告阳性 PC 的分析。

更新日期:2021-11-10
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