Shaw L, Williams R L, Hamill K J
Institute of Ageing and Chronic Disease, University of Liverpool, 6 West Derby Street, Liverpool, L78TX, UK.
BMC Res Notes. 2020 Feb 21;13(1):90. doi: 10.1186/s13104-020-04956-z.
The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LMβ1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models.
CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LMβ1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LMβ1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LMβ1 inhibits LM secretion, and is not a viable approach for use in animal models.
层粘连蛋白（LM）是一类基底膜糖蛋白，在支持上皮细胞、内皮细胞、神经和肌肉黏附以及调节包括细胞迁移、干细胞维持和分化在内的一系列过程中发挥着重要作用。然而，由于缺乏在必要分辨率下研究这些过程的合适工具，人们对LM网络的周转和重塑机制知之甚少。最近，人类LMβ1链的线虫秀丽隐杆线虫直系同源物在C端用可光转换荧光团Dendra2进行了标记。在此，我们利用基因组编辑在哺乳动物细胞系中建立了一个类似的系统，作为未来哺乳动物模型的概念验证。
利用CRISPR-Cas9在人肺腺癌细胞系A549的LMβ1的C端引入Dendra2序列。尽管标记蛋白在细胞内表达，但未检测到LMβ1-Dendra2蛋白沉积到编辑细胞的细胞外基质或条件培养基中。此外，编辑后的细胞增殖率降低。这些数据共同表明，在人类中，在LMβ1的C端添加Dendra2标签会抑制LM分泌，并且不是一种可用于动物模型的可行方法。
