Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2.
Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2.
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宿主胚胎发育生态位空缺是基于干细胞互补的器官再生（SCOG）所必需的。甲状腺转录因子1（TTF-1）是一种组织特异性转录因子，调节胚胎发育和分化的甲状腺，更重要的是，肺;因此，它已被认为是一个主基因敲除，以发展肺空缺主机。TTF-1基因敲除小鼠最初是通过基于胚胎干细胞的同源重组在基因的外显子3（E3 stop）中插入终止密码子而产生的。利用E3 stop宿主胚胎进行肺SCOG的主要问题是这些动物都有气管食管瘘（TEF），这不能通过供体干细胞来纠正，并且它们中的大多数具有单侧囊状肺。为了改进小鼠模型以实现基于SCOG的肺生成，在该项目中，我们使用CRISPR/Cas9工具通过受精卵显微注射去除基因的外显子2，并成功产生TTF-1敲除（E2 del）小鼠。与E3 stop相似，E2 del小鼠由于肺发育迟缓（具有囊状肺和仅发育不全的支气管树）、基底细胞增加但没有肺泡II型细胞和血管以及甲状腺发育异常而具有出生致死性。与E3 stop不同，57%的E2 del胚胎表现为I型气管发育不全（TA，一种人类先天性畸形），气管缩短，气管和食管清晰分离，而剩余的43%具有TEF。此外，所有E2 del小鼠均具有双侧囊状肺。SCOG中TA和双侧囊状肺均为首选。我们的工作提出了一种新的策略，用于生产可能对肺再生有用的SCOG宿主胚胎。
A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.
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影响因子：
16.6
作者：
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DOI：
10.1002/dvdy.24219
发表时间：
2015-03
期刊：
Developmental dynamics : an official publication of the American Association of Anatomists
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