Lins Jeremy, Hopkins Christopher E, Brock Trisha, Hart Anne C
Department of Neuroscience, Brown University, Providence, RI 02912.
InVivo Biosystems, Eugene, OR, 79402.
MicroPubl Biol. 2022 Sep 19;2022. doi: 10.17912/micropub.biology.000615. eCollection 2022.
To study important genes involved in Frontotemporal Dementia ( , and ), we created deletion alleles in the three orthologous genes ( , , and ). Simultaneously, we replaced the gene with the predicted orthologous human gene, often called whole-gene humanization, which allows direct assessment of conserved gene function, as well as the opportunity to examine consequences of clinical disease-associated patient variations. Each gene was manipulated using a different selection strategy, including a novel strategy using an mutation rescue technique. Clinical ALS/FTD missense variants G272V and P301L were successfully inserted in . Neither loss or clinical variants caused neuronal defects in young adult or aged , based on examination of glutamatergic phasmid neurons. Yet, we noted decreased survival to day 9 in the P301L strain, compared to control strains. Based on these results, we comment on strategies for humanization, including the importance of confirming gene predictions and identifying loss of function defects for each gene before embarking on humanization, and we report the creation of strains and a new gene-editing selection strategy that will be useful for future studies.
为了研究与额颞叶痴呆（ 、 和 ）相关的重要基因，我们在三个直系同源基因（ 、 和 ）中创建了缺失等位基因。同时，我们用预测的直系同源人类 基因替换了 基因，这通常被称为全基因人源化，它允许直接评估保守基因功能，也有机会研究临床疾病相关患者变异的后果。每个基因都使用不同的选择策略进行操作，包括一种使用 突变拯救技术的新策略。临床肌萎缩侧索硬化症/额颞叶痴呆错义变体G272V和P301L成功插入到 中。基于对谷氨酸能噬菌粒神经元的检查，无论是 缺失还是临床变体在年轻成年或老年 中均未引起神经元缺陷。然而，与对照菌株相比，我们注意到P301L 菌株在第9天的存活率下降。基于这些结果，我们评论了人源化策略，包括在进行人源化之前确认 基因预测以及识别每个基因功能缺失缺陷的重要性，并且我们报告了菌株的创建和一种新的基因编辑选择策略，这将对未来的研究有用。
