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用于人干细胞条件表达的 Lent-On-Plus Lentiviral 载体。

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

机构信息

Genomic Medicine Department. GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Goverment, Parque Tecnológico Ciencias de la Salud, Av. de la Ilustración 114, 18016 Granada, Spain.

LentiStem Biotech. GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Goverment, Parque Tecnológico Ciencias de la Salud, Av. de la Ilustración 114, 18016 Granada, Spain.

出版信息

Sci Rep. 2016 Nov 17;6:37289. doi: 10.1038/srep37289.

DOI:10.1038/srep37289
PMID:27853296
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5112523/
Abstract

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

摘要

由于现有的传递方法效率低下、转基因沉默严重以及调节剂毒性等问题，人类干细胞中的条件性转基因表达一直难以实现。大多数现有的技术都是基于表达适当水平的 tTA 或 rtTA 转录激活物的干细胞克隆（基于 TetR-VP16 嵌合体）。在本研究中，我们旨在生成 Tet-On 全合一慢病毒载体（LVs），利用原始 TetR 抑制剂在人类干细胞中严格调控转基因表达。通过使用适当的启动子组合并利用 Is2 绝缘子屏蔽 LVs，我们构建了 Lent-On-Plus Tet-On 系统，该系统在人类多能和多能干细胞中实现了高效的转基因调控。使用 Lent-ON-Plus LVs 生成诱导性干细胞系不需要选择或克隆，并且在长期培养和向不同谱系分化后，转基因调控仍能维持。据我们所知， Lent-On-Plus 是第一个在人类多能干细胞及其后代的大量群体中严格调控转基因表达的全合一载体系统。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/f039235597bb/srep37289-f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/c2e3bef0eca3/srep37289-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/60aa07db21f8/srep37289-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/2b3fd935266f/srep37289-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/4f2a5d890432/srep37289-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/4a9f00a5f5b8/srep37289-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/cf1c47113933/srep37289-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/f039235597bb/srep37289-f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/c2e3bef0eca3/srep37289-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/60aa07db21f8/srep37289-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/2b3fd935266f/srep37289-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/4f2a5d890432/srep37289-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/4a9f00a5f5b8/srep37289-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/cf1c47113933/srep37289-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/51c8/5112523/f039235597bb/srep37289-f7.jpg

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