Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
PLoS One. 2018 Jun 28;13(6):e0199785. doi: 10.1371/journal.pone.0199785. eCollection 2018.
FAcilitates Chromatin Transcription (FACT) is a complex of SSRP1 and SPT16 that is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT has been mostly studied in cell-free or single cell model systems because general FACT knockout (KO) is embryonically lethal (E3.5). FACT levels are limited to the early stages of development and stem cell niches of adult tissues. FACT is upregulated in poorly differentiated aggressive tumors. Importantly, FACT inhibition (RNAi) is lethal for tumors but not normal cells, making FACT a lucrative target for anticancer therapy. To develop a better understanding of FACT function in the context of the mammalian organism under normal physiological conditions and in disease, we aimed to generate a conditional FACT KO mouse model. Because SPT16 stability is dependent on the SSRP1-SPT16 association and the presence of SSRP1 mRNA, we targeted the Ssrp1 gene using a CreERT2- LoxP approach to generate the FACT KO model. Here, we highlight the limitations of the CreERT2-LoxP (Rosa26) system that we encountered during the generation of this model. In vitro studies showed an inefficient excision rate of ectopically expressed CreERT2 (retroviral CreERT2) in fibroblasts with homozygous floxed Ssrp1. In vitro and in vivo studies showed that the excision efficiency could only be increased with germline expression of two alleles of Rosa26CreERT2. The expression of one germline Rosa26CreERT2 allele led to the incomplete excision of Ssrp1. The limited efficiency of the CreERT2-LoxP system may be sufficient for studies involving the deletion of genes that interfere with cell growth or viability due to the positive selection of the phenotype. However, it may not be sufficient for studies that involve the deletion of genes supporting growth, or those crucial for development. Although CreERT2-LoxP is broadly used, it has limitations that have not been widely discussed. This paper aims to encourage such discussions.
参与染色质重塑的复合物 FACT 由 SSRP1 和 SPT16 组成，在转录、复制和 DNA 修复过程中参与染色质重塑。由于普遍的 FACT 敲除（KO）在胚胎期是致命的（E3.5），因此 FACT 主要在无细胞或单细胞模型系统中进行研究。FACT 的水平仅限于发育的早期阶段和成年组织的干细胞龛。FACT 在分化不良的侵袭性肿瘤中上调。重要的是，FACT 抑制（RNAi）对肿瘤是致命的，但对正常细胞没有影响，这使得 FACT 成为癌症治疗的一个有吸引力的靶点。为了在正常生理条件和疾病下更好地了解哺乳动物体内 FACT 的功能，我们旨在生成一种条件性 FACT KO 小鼠模型。由于 SPT16 的稳定性依赖于 SSRP1-SPT16 的结合和 SSRP1 mRNA 的存在，我们使用 CreERT2-LoxP 方法靶向 Ssrp1 基因，生成 FACT KO 模型。在这里，我们强调了在生成这种模型时遇到的 CreERT2-LoxP（Rosa26）系统的局限性。体外研究表明，在同源纯合 floxed Ssrp1 的成纤维细胞中，异位表达的 CreERT2（逆转录病毒 CreERT2）的切除率效率低下。体外和体内研究表明，只有通过生殖系表达 Rosa26CreERT2 的两个等位基因，才能提高切除效率。一个生殖系 Rosa26CreERT2 等位基因的表达导致 Ssrp1 的不完全切除。CreERT2-LoxP 系统的效率有限，对于涉及由于表型的正选择而干扰细胞生长或活力的基因缺失的研究可能是足够的。然而，对于涉及支持生长或对发育至关重要的基因缺失的研究可能是不够的。尽管 CreERT2-LoxP 被广泛使用，但它存在尚未广泛讨论的局限性。本文旨在鼓励此类讨论。
