Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.
Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.
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DOI：
10.3389/fcell.2022.914386
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发表时间：
2022
影响因子：
5.5
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视网膜Müller神经胶质细胞的直接重编程是替代因视网膜营养不良而丢失的光感受器和视网膜神经节细胞的有希望的途径。然而，最近提出的问题，声称有效的胶质细胞到神经元的视网膜重编程的研究，使用GFAP迷你启动子驱动的腺相关病毒（AAV）载体进行的准确性。在这项研究中，我们已经解决了这些问题，使用GFAP迷你启动子驱动的AAV构建体，同时过表达mCherry报告和候选转录因子预测诱导神经胶质细胞到神经元的转换，结合视网膜Müller神经胶质细胞的前瞻性遗传标记使用诱导型Cre依赖的GFP报告。我们发现，虽然对照GFAP-mCherry构建体在Müller胶质细胞中忠实表达，但测试的7种转录因子过表达构建体中有5种主要在无长突细胞和视网膜神经节细胞中表达。这些发现证明了对基于AAV的GFAP迷你启动子特异性的强插入依赖性作用，这排除了其在研究视网膜中的胶质细胞到神经元转化时推断细胞谱系关系的用途。
Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.