CRISPR/Cas9 介导的 GFP-人牙本质基质蛋白 1（DMP1）启动子在间充质干细胞 ROSA26 基因座的基因敲入用于监测成骨细胞分化。

CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.

机构信息

National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.

Targeted Drug Delivery Research Center, Institute of Pharmaceutical Technology, Mashhad University of Medical Sciences, Mashhad, Iran.

出版信息

J Gene Med. 2020 Dec;22(12):e3288. doi: 10.1002/jgm.3288. Epub 2020 Oct 29.

DOI:10.1002/jgm.3288

PMID:33047833

Abstract

BACKGROUND

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation.

METHODS

In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays.

RESULTS

We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs .

CONCLUSIONS

The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.

摘要

背景

牙本质基质蛋白 1（DMP1）在矿化牙和骨中高度表达，在矿化和磷酸盐代谢中发挥关键作用。DMP1 在成骨前体细胞核中的一个重要表达作用是上调骨钙蛋白和碱性磷酸酶等成骨细胞特异性基因。本研究旨在探讨人 DMP1 启动子作为成骨细胞分化指示标记物的潜在应用。

方法

本研究通过 CRISPR/Cas9 系统构建了 DMP1 启动子-DsRed-GFP 敲入间充质干细胞（MSCs），该系统可自动检测成骨细胞分化。应用同源定向敲入策略，将 DMP1 启动子的 2kb 片段插入 GFP 和 DsRed 报告基因盒的上游，整合到人 ROSA 基因座，生成双荧光细胞。我们进一步在成骨培养基中分化 MSCs，以监测 MSCs 的命运。首先，使用 CRISPR/Cas9 质粒转染细胞，导致 MSCs 具有绿色荧光强度，然后使用嘌呤霉素选择 GFP 阳性细胞。其次，将 GFP 阳性 MSCs 分化为成骨细胞，其红色荧光强度增加。通过进行碱性磷酸酶和茜素红染色来验证 MSCs 的成骨分化。