CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration.
CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration.
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DOI：
10.1016/j.stemcr.2014.06.018
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发表时间：
2014-09-09
影响因子：
5.9
通讯作者：
Tanaka, Elly M.
中科院分区：
文献类型：
--
作者：
Fei, Ji-Feng;Schuez, Maritta;Tazaki, Akira;Taniguchi, Yuka;Roensch, Kathleen;Tanaka, Elly M.
蝾螈是唯一能够再生肢体和脊髓（SC）所有细胞类型的四足动物，因此代表了一种重要的再生模型，但缺乏基因敲除技术限制了分子分析。我们比较了转录激活因子样效应核酸酶（TALENs）和聚集规律间隔短回文重复序列（crispr）在敲除美西螈三个基因座中的作用，发现与TALENs相比，crispr具有高渗透性敲除，毒性作用更小。在高达100%的细胞中缺失Sox2，产生存活的F0幼虫，SC组织正常，室管膜胶质细胞标记物如GFAP和ZO-1表达正常。然而，在断尾后，神经干细胞增殖受到抑制，导致脊髓特异性再生失败。而中胚芽形成正常。在发育过程中表达Sox3，而不是再生，很可能允许胚胎存活和再生特异性表型。这一分析代表了第一个组织特异性再生表型从一个基因在蝾螈基因组缺失。在蝾螈中，crispr比TALENs产生更有效的基因敲除。Sox2-CRISPR蝾螈幼虫具有正常的脊髓，但表现出再生表型。在Sox2-CRISPR蝾螈的再生过程中缺乏神经干细胞的扩增。Tanaka和他的同事表明，crispr可以有效敲除蝾螈中的靶基因，而毒性比TALENs小。crispr介导的Sox2基因敲除导致脊髓发育正常，但由于神经干细胞增殖减少，脊髓再生失败。这项工作代表了蝾螈基因组基因缺失后的第一个组织特异性再生表型。
The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC) and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl. CRISPRs yield more efficient and effective gene knockout than TALENs in axolotls Sox2-CRISPR axolotl larvae have a normal spinal cord but show a regeneration phenotype There is a lack of neural stem cell expansion during regeneration in Sox2-CRISPR axolotls Tanaka and colleagues show that CRISPRs efficiently knock out target genes in the axolotl with less toxicity than TALENs. CRISPR-mediated Sox2 knockout resulted in normal spinal cord development, but spinal cord regeneration failure due to reduced proliferation of neural stem cells. This work represents the first tissue-specific regeneration phenotype upon genomic deletion of a gene in the axolotl.
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