Characterization of novel endothelial-specific and smooth-muscle specific Poldip2 knockout mice

Poldip2 is a multifunctional protein whose roles are only partially understood. The previous Poldip2 knockout mouse generated by the gene trap model suffers from two limitations: perinatal lethality in homozygotes and constitutive Poldip2 inactivation. To overcome these limitations, we developed a new conditional floxed Poldip2 mouse. Here, we correctly targeted floxed Poldip2 mice to produce an endothelial-specific by crossing it with CDH5 ERT2 Cre mouse. Additionally, we also produced smooth-muscle specific Poldip2 knockout mice by crossing the flox'ed Poldip2 mice with SM22A-Cre mice. The Poldip2_EC-/- and Poldip2_SMC-/-mice were viable and showed remarkably reduced Poldip2 expression. To characterize the effect of Poldip2 ablation in the ECs and in the SMCs, we performed a RNA-seq study using the carotid arteries of these mice. The carotid arteries were harvested from the Poldip2 knockout mice (n=4) as well as from the littermate controls (n=4). The lumen of these arteries were quickly flushed with Qiazol to extract the endothelial-enriched RNAs. Also, RNAs were extracted from the leftover arteries (containing the media and adventitia, consisting of smooth muscle cells and other adventiatial cells). These RNAs were subjected to RNAseq to determine the effect of Poldip2 knockdown in endothelium and in the vascular smooth muscle fraction of the carotid arteries.Poldip2是一种多功能蛋白质，其生物学功能仅得到部分阐明。此前通过基因陷阱模型构建的Poldip2敲除小鼠存在两大局限：纯合子个体出现围产期致死，且为组成型Poldip2基因失活。为克服上述局限，我们研发了一种新型条件性floxed Poldip2小鼠。本研究中，我们通过将该floxed Poldip2小鼠与CDH5 ERT2 Cre小鼠杂交，成功构建了内皮细胞特异性Poldip2敲除小鼠；此外，我们还通过将floxed Poldip2小鼠与SM22A-Cre小鼠杂交，获得了平滑肌细胞特异性Poldip2敲除小鼠。Poldip2_EC-/-与Poldip2_SMC-/-小鼠均可正常存活，且Poldip2的表达水平显著降低。为解析Poldip2在血管内皮细胞（ECs）和平滑肌细胞（SMCs）中的敲除效应，我们利用上述小鼠的颈动脉组织开展了RNA测序（RNA-seq）研究。我们分别采集了每组n=4的Poldip2特异性敲除小鼠及其同窝对照小鼠的颈动脉组织，将每根颈动脉的管腔用Qiazol试剂快速冲洗以提取富集内皮细胞的RNA，同时从剩余的血管组织（包含中膜与外膜，由平滑肌细胞及其他外膜细胞构成）中提取总RNA，随后将上述两类RNA样本进行RNA-seq测序，以明确Poldip2在颈动脉内皮细胞及血管平滑肌组分中的敲除调控效应。