Supplement to EY021482 to carry out a screen for retinal regeneration using CRISPR-Cas9 gene activation.

描述（由申请人提供）：与神经系统的其他区域一样，视网膜易患许多获得性和遗传性神经元退行性疾病。由于视网膜为大脑提供所有视觉感官信息的输入，因此细胞的损失导致视觉障碍和潜在的完全失明。许多视网膜变性疾病仅影响视网膜细胞的子集，尽管在更晚期的疾病中，经常会发生整个视网膜的损失和重组。在哺乳动物中，退化细胞的再生非常有限;然而，在鱼类中，视网膜损伤后，所有类型的新神经元都从M？ller胶质细胞中再生，并且它们在功能上整合到现有的电路中。然而，M？ller神经胶质细胞，再生的细胞来源，存在于所有脊椎动物的视网膜中。在我们三年前提交的提案中，我们假设哺乳动物M？ller胶质细胞的再生是有限的，因为它们在损伤后不能表达一种关键的前神经转录因子Ascl 1。我们建议通过病毒介导的Ascl 1在小鼠M？ller神经胶质细胞中的表达来验证这一假设。在两年的资助中，我们已经验证了这一假设，并发现Ascl 1的病毒表达足以激活小鼠M？ller胶质细胞中的神经原性程序，无论是在分离的培养物中还是在完整的视网膜中。重新编程的米勒神经胶质细胞产生的细胞在形态、基因表达和对神经递质的反应方面与神经元相似。在下一个资助期内，我们建议使用其他转录因子和表观遗传修饰剂进一步优化这种重编程过程，然后测试Ascl 1重编程的Muller胶质细胞是否可以在新开发的转基因小鼠系中提供体内再生来源。

DESCRIPTION (provided by applicant): Like other areas of the nervous system, the retina is subject to many acquired and inherited neuronal degenerative diseases. Since the retina provides the input for all visual sensory information to the brain, the loss of cells results in viual impairment and potentially complete blindness. Many retinal degenerative diseases affect only a subset of the retinal cells, although, frequently in more advanced disease, loss and reorganization of the entire retina can occur. In mammals, there is very limited regeneration of the degenerated cells; however, in fish, new neurons of all types regenerate from M¿ller glia following retinal damage and they are functionally integrated into the existing circuitry. Nevertheless, M¿ller glia, the cellular source for regeneration, is present in all vertebrate retins. In the proposal we submitted three years ago, we hypothesized that regeneration from mammalian M¿ller glia was limited because they fail to express a key proneural transcription factor, Ascl1, after injury. We proposed to test this hypothesis by virally-mediated expression of Ascl1 in mouse M¿ller glia. In the two years of funding, we have tested the hypothesis, and found that viral expression of Ascl1 is sufficient to activate a neurogenic program in mouse M¿ller glia, both in dissociated cultures and in the intact retina. The reprogrammed M¿ller glia generates cells that resemble neurons in morphology, gene expression and their responses to neurotransmitters. In the next funding period, we propose to further optimize this reprogramming process, using other transcription factors and epigenetic modifiers, and then to test whether Ascl1-reprogrammed Muller glia can provide a source for regeneration in vivo in a newly developed line of transgenic mice.