Abstract
Abstract in English, Chinese In an attempt to generate g.A746G substitution in the BMPR-IB gene, we unexpectedly obtained BMPR-IB homozygous knockout piglets ( BMPR-IB -/-) and heterogeneous knockout piglets with one copy of the A746G mutation ( BMPR-IB -/746G) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region. All BMPR-IB-disrupted piglets showed an inability to stand and walk normally. Both BMPR-IB -/- and BMPR-IB -/746G piglets exhibited severe skeletal dysplasia characterized by distorted and truncated forearms (ulna, radius) and disordered carpal, metacarpal, and phalangeal bones in the forelimbs. The piglets displayed more severe deformities in the hindlimbs by visual inspection, including fibular hemimelia, enlarged tarsal bone, and disordered toe joint bones. Limb deformities were more profound in BMPR-IB -/- piglets than in the BMPR-IB -/746G piglets. Proteomic analysis identified 139 differentially expressed proteins (DEPs) in the hindlimb fibula of BMPR -IB -/746G piglets compared to the wild-type (WT) controls. Most DEPs are involved in skeletal or embryonic development and/or the TGF-β pathway and tumor progression. Gene Ontology (GO) and protein domain enrichment analysis suggested alterations in these processes. Of the top 50 DEPs, a large proportion, e.g., C1QA, MYO1H, SRSF1, P3H1, GJA1, TCOF1, RBM10, SPP2, MMP13, and PHAX, were significantly associated with skeletal development. Our study provides novel findings on the role of BMPR-IB in mammalian limb development. 在之前的研究中，我们原本计划通过CRISPR/Cas9编辑技术在猪的 BMPR-IB基因中产生g.A746G的突变，然而却意外地获得了 BMPR-IB纯合敲除仔猪（ BMPR-IB -/-）和携带A746G突变的杂合子敲除仔猪（ BMPR-IB -/746G）。PCR检测及测序结果显示 BMPR-IB基因区域中发生了复杂的基因组重排。所有 BMPR-IB -/-和 BMPR-IB -/746G仔猪都无法正常站立和行走。解剖学观察发现，这些仔猪均呈现出严重的四肢骨骼发育异常，其中前肢的前臂骨（尺骨、桡骨）扭曲及截短，腕骨、掌骨和指骨排列紊乱；相比前肢，后肢表现出更严重的畸形，包括腓侧半肢畸形、跗骨增大和脚趾关节骨排列紊乱。与 BMPR-IB -/746G个体相比， BMPR-IB -/-仔猪的四肢骨骼畸形更为严重。通过蛋白质组学分析，在杂合子敲除仔猪（ BMPR-IB -/746G）和对照组仔猪（WT）的后肢腓骨中鉴别到139个差异表达蛋白（DEPs），大多数DEPs是已知的参与骨骼或胚胎发育、TGF-β通路或肿瘤进展的重要因子；基因本体（GO）和蛋白质结构域富集分析也显示出这些通路上的变化。在排列前50的DEPs中，有一大部分蛋白如C1QA、MYO1H、SRSF1、P3H1、GJA1、TCOF1、RBM10、SPP2、MMP13及PHAX，均已被证明与骨骼发育显著相关。该研究为解析 BMPR-IB在哺乳动物肢体发育中的作用提供了新的认识。.