Sano Soichi, Walsh Kenneth
Laboratory of Cardiovascular Mosaicism, National Cerebral and Cardiovascular Center, Osaka, Japan.
Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA, USA.
Bio Protoc. 2023 Aug 5;13(15):e4729. doi: 10.21769/BioProtoc.4729.
This protocol describes the generation of chimeric mice in which the Y chromosome is deleted from a proportion of blood cells. This model recapitulates the phenomenon of hematopoietic mosaic loss of Y chromosome (mLOY), which is frequently observed in the blood of aged men. To construct mice with hematopoietic Y chromosome loss, lineage-negative cells are isolated from the bone marrow of ROSA26-Cas9 knock-in mice. These cells are transduced with a lentivirus vector encoding a guide RNA (gRNA) that targets multiple repeats of the Y chromosome centromere, effectively removing the Y chromosome. These cells are then transplanted into lethally irradiated wildtype C57BL6 mice. Control gRNAs are designed to target either no specific region or the fourth intron of gene. Transduced cells are tracked by measuring the fraction of blood cells expressing the virally encoded reporter gene tRFP. This model represents a clinically relevant model of hematopoietic mosaic loss of Y chromosome, which can be used to study the impact of mLOY on various age-related diseases. Graphical overview.
本方案描述了嵌合小鼠的生成过程，其中一部分血细胞的Y染色体被删除。该模型重现了造血Y染色体镶嵌性缺失（mLOY）现象，这在老年男性血液中经常观察到。为构建具有造血Y染色体缺失的小鼠，从ROSA26-Cas9基因敲入小鼠的骨髓中分离出谱系阴性细胞。用编码靶向Y染色体着丝粒多个重复序列的引导RNA（gRNA）的慢病毒载体转导这些细胞，从而有效去除Y染色体。然后将这些细胞移植到经致死性照射的野生型C57BL6小鼠体内。对照gRNA设计为不靶向任何特定区域或靶向基因的第四内含子。通过测量表达病毒编码报告基因tRFP的血细胞比例来追踪转导细胞。该模型代表了造血Y染色体镶嵌性缺失的临床相关模型，可用于研究mLOY对各种与年龄相关疾病的影响。图形概述。
