Glycosylation as a tracer of off-target Cre-lox activation in development.
Glycosylation as a tracer of off-target Cre-lox activation in development.
复制标题
糖基化作为发育过程中脱靶 Cre-lox 激活的示踪剂。
DOI：
10.1093/glycob/cwae023
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发表时间：
2024
期刊：
影响因子：
4.3
通讯作者：
Cobb,BrianA
中科院分区：
文献类型：
--
作者：
Glendenning,LeandreM;Reynero,KalobM;Cobb,BrianA
Cre-lox系统是用于体内谱系特异性和诱导型基因组编辑的最广泛使用的方法之一。然而，由于在干细胞或多能前体细胞中瞬时启动子表达引起的不完全转移和脱靶效应可能是有问题的并且难以检测，特别是如果靶基因通常不存在于完全分化但脱靶的细胞中。然而，通过Cre的瞬时表达导致的靶基因的丢失可能会影响那些细胞由于在前体群体中的瞬时表达而分化。在这些情况下，未知前体细胞中的脱靶效应充其量会使从模型中得出的结论复杂化，最坏的情况是使从该敲除菌株产生的所有数据无效。因此，鉴定沿着整个细胞谱系的Cre-driver启动子表达对于提高严谨性和再现性至关重要。作为一个例子，在早期前体细胞中的瞬时表达已经在多种Cre菌株中被记录，例如用作“内皮细胞特异性”模型的基于Tie 2的Cre驱动系统1。然而，现在已知Tie 2在造血和内皮细胞谱系的干细胞上游瞬时表达。在这里，我们使用Tie 2 Cre驱动菌株来证明，由于其普遍存在的性质，质膜聚糖是基于Cre的敲除的特异性和特异性的有用标记。
The Cre-loxsystem is one of the most widely used methods for lineage-specific and inducible genome editing in vivo. However, incomplete penetrance and off-target effects due to transient promoter expression in a stem or pluripotent precursor cell can be problematic and difficult to detect, especially if the target gene is not normally present in the fully differentiated but off-target cells. Yet, the loss of the target gene through the transient expression of Cre may impact the differentiation of those cells by virtue of transient expression in a precursor population. In these situations, off-target effects in an unknown precursor cell can, at best, complicate conclusions drawn from the model, and at worst, invalidate all data generated from that knockout strain. Thus, identifying Cre-driver promoter expression along entire cell lineages is crucial to improve rigor and reproducibility. As an example, transient expression in an early precursor cell has been documented in a variety of Cre strains such as the Tie2-based Cre-driver system that is used as an “endothelial cell-specific” model1. Yet, Tie2 is now known to be transiently expressed in a stem cell upstream of both hematopoietic and endothelial cell lineages. Here, we use the Tie2 Cre-driver strain to demonstrate that due to its ubiquitous nature, plasma membrane glycans are a useful marker of both penetrance and specificity of a Cre-based knockout.