BioVector® Mouse Piezo1 Overexpression Plasmid / 鼠源 Piezo1 过表达质粒

一 产品基本信息与遗传学背景

• 质粒名称：鼠源 Piezo1 过表达质粒（Mouse Piezo1 Overexpression Plasmid）。

• 目的基因背景（Piezo1）：

  • 基因别名：Piezo1, Fam38a。

  • 物种来源：小鼠（Mus musculus）。

  • 蛋白结构与功能：Piezo1 是一种进化上高度保守的由机械力激活的非选择性阳离子通道（Mechanosensitive Ion Channel）。小鼠 Piezo1 单体极其庞大，由多达 2547 个氨基酸残基组成，包含多达 38 个跨膜螺旋区域。在天然状态下，三个 Piezo1 单体组装成一个独特的、形似“三叶螺旋桨”的同源三聚体结构，在细胞膜表面形成一个向内凹陷的巨大穹窿或“纳米圆顶”（Nano-dome）。

  • 通透性特性：当细胞膜受到机械刺激（如流体剪切力、渗透压改变、牵张拉伸或牵引力）时，Piezo1 通道会迅速开放，主要介导 $Ca^{2+}$（钙离子）的快速内流，同时通透 $Na^+$、$K^+$ 等单价阳离子。这能将物理机械信号瞬时转译为细胞内的生物化学信号（如激活钙信号通路、NF-AT、FAK 等下游级联）。

• 载体构件特征：

  • 强启动子：目的基因通常克隆于由 CMV 或 EF1a 强启动子驱动的哺乳动物高效表达载体（如 pCDNA3.1(+)、pEGFP-N1 或 pLenti 慢病毒载体）中，以确保在宿主细胞内实现高水平的结构性过表达。

  • 标签/荧光蛋白融合（依据具体批次变体）：可选融合 FLAG、HA、Myc 等小肽标签，或在 C 端/N 端融合 GFP/mCherry 荧光蛋白，便于后期通过 Western Blot（WB）、免疫共沉淀（Co-IP）或活细胞荧光成像追踪 Piezo1 在细胞膜上的空间定位。

• 抗性筛选标记：

  • 大肠杆菌抗性：氨苄青霉素抗性（Ampicillin, AmpR） 或 卡那霉素抗性（Kanamycin, KanR），用于工程菌扩增。

  • 哺乳动物细胞筛选抗性：通常带有 新霉素（G418/Neomycin）、嘌呤霉素（Puromycin）或潮霉素（Hygromycin B）抗性标记。

二 核心科研价值与转化医学应用

鼠源 Piezo1 过表达质粒是开展生物力学（Mechanobiology）和血管/骨骼发育等前沿领域研究的基石工具：

1. 细胞机械感应与物理力转导研究（Mechanotransduction）：

   用于深入探究细胞如何感知周围微环境的硬度或外界压力。过表达 Piezo1 后，细胞对流体剪切力（Fluid Shear Stress）或细胞划痕拉伸的敏感度大幅上调。常用于研究血管内皮细胞在血流剪切力下的重塑、成骨细胞在重力/机械载荷下的骨形成，以及免疫细胞（如巨噬细胞）在硬质基质中的激活机制。

2. 细胞钙信号动力学监测（Calcium Signaling Kinetics）：

   通过将该质粒转染至易于操作的工具细胞（如 HEK293T）中，配合 Fluo-4 AM 等钙离子荧光探针或高敏感钙成像系统，可体外重现由物理压迫或 Piezo1 特异性小分子激动剂（如 Yoda1, Jedi1/2）诱导的特征性钙内流脉冲，用于验证该通道的电生理活性。

3. 组织器官发育与病理机制建模：

   小鼠 Piezo1 在红细胞体积调节、血管新生、淋巴管发育以及上皮细胞稳态维持中扮演关键角色。利用该质粒在特定原代细胞中实施过表达，可用于模拟和研究剪切力敏感性遗传性干瘪红细胞增多症（Hereditary Xerocytosis）、高血压导致的血管壁肥厚、以及肿瘤微环境硬度改变引发的癌细胞迁移和侵袭。

三 实验室质粒转化、扩增、高纯度提取与转染标准步骤

1. 扩增菌株与培养基配置

由于 Piezo1 的编码序列（CDS）长达 7.6 kb 左右，整包质粒通常超过 12-14 kb，属于巨大的大质粒，普通质粒常规扩增极易发生自发性同源重组导致目的片段缺失。

• 推荐大肠杆菌宿主：严禁使用常规 DH5a 扩增。强烈推荐选用专为大质粒、不稳定性重复序列设计的 Stbl3 或 SURE 感受态细胞。

• 培养基与抗性配置：LB 肉汤/固体琼脂，添加最终工作浓度为 100 ug/mL 氨苄青霉素（Ampicillin，或依说明书改用 50 ug/mL 卡那霉素）。为了防止大质粒重组，建议将摇菌扩增温度由常规的 37 摄氏度降低至 30 摄氏度过夜慢摇。

2. 大肠杆菌转化与复苏步骤

1. 取出 50 - 100 uL 的大肠杆菌 Stbl3 感受态细胞置于冰上缓慢融化。

2. 加入 1 - 2 uL 鼠源 Piezo1 过表达质粒 DNA，轻弹管底混匀（严禁涡旋震荡），冰浴 30 分钟。

3. 将离心管迅速置于 42 摄氏度水浴中精确热击 45 秒，随后立即插回冰中静置 2 分钟，切勿摇动。

4. 向管内加入 500 uL 不含抗生素的无菌 LB 肉汤（或 SOC 培养基），置于 30 摄氏度（或 37 摄氏度）振荡培养箱内，以 200 rpm 复苏匀速摇菌 60 分钟。

5. 4000 rpm 离心 3 分钟，弃去部分上清，留约 100 uL 液体将菌体吹匀，平铺涂布于含有对应抗性的 LB 固体平板上。

6. 置于 30 摄氏度（建议）培养箱中倒置培养 16 - 20 小时（若在 30 摄氏度生长较慢，可能需要更长时间，直至长出清晰可见的健壮单菌落）。

3. 质粒扩增与无内毒素提取（Midiprep / Maxiprep）

1. 挑取平板上边缘清晰的单个单菌落，接种至 5 mL 含抗生素的 LB 液体肉汤试管中，30 摄氏度、220 rpm 预培养 6 - 8 小时。

2. 随后按 1:500 比例扩增接种至 100 - 200 mL 含抗生素的液体 LB 培养基中，置于 30 摄氏度 连续振荡过夜培养 14 - 16 小时。

3. 提取关键：由于过表达质粒后续直接用于哺乳动物细胞转染，且 Piezo1 对细胞状态极为敏感，必须使用无内毒素的质粒中提/大提试剂盒（Endotoxin-free Plasmid Kit）。残留的内毒素会严重干扰细胞对机械力的感应并引发非特异性钙内流。

4. 提取出的质粒溶解于无核酸酶的灭菌水中，利用分光光度计测量浓度。对于超大质粒，建议抽样 1 uL 进行常规琼脂糖凝胶电泳（或测序鉴定），确保带型单一、无降解、无条带缺失。

4. 哺乳动物细胞转染（Transient Transfection）

以常规 HEK293T 或小鼠原代内皮细胞在 T25 瓶（或 6 孔板）中的操作为例：

1. 细胞密度：转染前一天接种细胞，确保在转染当天细胞密度达到 60% - 70% 融合度。

2. 转染体系（以 6 孔板单孔为例）：

   • A管：将 2 - 2.5 ug 的高质量纯化小鼠 Piezo1 质粒 DNA 稀释于 100 uL 无血清的 Opti-MEM 培养基中，轻柔混匀。

   • B管：将 4 - 6 uL 脂质体转染试剂（如 Lipofectamine 2000 或 3000）稀释于 100 uL 无血清的 Opti-MEM 中，混匀并静置 5 分钟。

3. 复合物组装：将 A 管液体全部滴入 B 管中，轻弹混匀，在室温下静置 15 - 20 分钟以形成稳定的质粒-脂质体复合物。

4. 侵染：吸除细胞旧培养基，更换为新鲜的低血清或完全培养基（根据转染试剂说明）。将复合物均匀滴加至孔内，轻微前后晃动。

5. 维持与检测：置于 37 摄氏度、5% CO2 孵箱中培养 4 - 6 小时后更换为新鲜的完全培养基。转染 24 - 48 小时后，Piezo1 蛋白在细胞膜表面达到表达峰值。此时可施加 Yoda1 激动剂、流体剪切力或划痕刺激，通过 Western Blot、免疫荧光或活细胞钙成像（Calcium Imaging）检测其功能性过表达指标。

Part 2 English Section

I General Information and Genetic Architecture

• Plasmid Designation: Mouse Piezo1 Overexpression Plasmid.

• Target Gene Architecture (Mouse Piezo1 Matrix):

  • Gene Aliases: Piezo1, Fam38a.

  • Species Origin: Mouse (Mus musculus).

  • Protein Topology & Function: Piezo1 functions as an evolutionarily conserved, mechanically activated non-selective cation channel. The mouse Piezo1 monomer is exceptionally massive, consisting of 2,547 amino acid residues organized into 38 transmembrane helices. Homotrimeric assembly of three separate monomers forms a highly unique, curved "three-bladed propeller" macro-structure, indenting the plasma membrane to create an inward-facing "nano-dome".

  • Permeability Matrix: Physical deformation of the lipid bilayer (e.g., via fluid shear stress, osmotic pressure shifts, cell membrane stretching, or intracellular traction forces) rapidly gates open the channel pore, preferentially mediating a massive influx of $Ca^{2+}$ (calcium ions) alongside single-valence cations ($Na^+$, $K^+$). This translates tactile mechanical events into long-range intracellular biochemical signals (e.g., activating downstream FAK, NF-AT, and general calcium-dependent signaling networks).

• Vector Backbone Engineering Modules:

  • Strong Promoter: The definitive protein-coding sequence (CDS) is systematically cloned downstream of hyper-active promoters (such as CMV or EF1a) inside mammalian expression plasmids (e.g., pCDNA3.1(+), pEGFP-N1, or pLenti lentiviral arrays) to ensure robust constitutive overexpression in target eukaryotic lineages.

  • Tagging/Fluorescent Options (Batch Dependent Variants): Features optional small peptide fusions (e.g., FLAG, HA, Myc) or C-/N-terminal Green Fluorescent Protein (GFP) or mCherry tags, enabling precise localization tracking at the lipid bilayer via Western Blotting (WB), Co-Immunoprecipitation (Co-IP), or live-cell confocal laser scanning imaging.

• Selective Resistance Elements:

  • Bacterial Selective Pressure: Ampicillin resistance (AmpR) or Kanamycin resistance (KanR) for high-yield plasmid propagation.

  • Eukaryotic Selection Cascades: Typically driven by Neomycin (G418), Puromycin, or Hygromycin B resistance vectors.

II Strategic Research Value and Translational Fields

The mouse Piezo1 overexpression plasmid serves as a foundational bio-tactile tool within mechanobiology, tissue morphogenesis, and vascular physiology:

1. Mechanotransduction Dynamics & Matrix Rigidity Sensing:

   Deployed to dissect how somatic cells map the stiffness of the extracellular matrix (ECM) or respond to macro-environmental pressures. Overexpressing Piezo1 drastically up-regulates cellular sensitivity toward shear forces, scratch-induced mechanical deformation, and stretch matrices. This models how vascular endothelial cells react to hemodynamic shear stress, how osteoblasts drive osteogenesis under load conditions, and how immune cells (e.g., macrophages) polarize within fibrotic, rigid tissue niches.

2. Kinetics of Intracellular Calcium Flux Analysis:

   By transfecting this plasmid into robust model systems (e.g., HEK293T lines), investigators can load monolayers with premium calcium-responsive indicators (such as Fluo-4 AM). Upon delivery of specialized physical pressures or low-molecular-weight chemical agonists (such as Yoda1, Jedi1/2), custom imaging channels record massive, reproducible $Ca^{2+}$ ion influx kinetics, benchmarking ion channel pore activity.

3. Modeling Organ Development and Mechanical Pathology:

   Endogenous mouse Piezo1 dictates erythrocyte volume homeostasis, angiogenesis, lymphatic patterning, and epithelial cellular crowding loops. Utilizing localized transient or stable overexpression maps how functional gains contribute to conditions like dehydration-mediated Hereditary Xerocytosis, shear-stress induced arterial wall hypertrophy, and matrix-stiffness driven cancer cell epithelial-mesenchymal transition (EMT) and metastatic invasion.

III Bacterial Transformation, Proliferation, Ultrapure Extraction, and Mammalian Transfection Routines

Because the mouse Piezo1 CDS expands across ~7.6 kb, the entire plasmid structure routinely eclipses 12–14 kb. Such massive constructs are highly prone to spontaneous homologous recombination and large-scale insert deletions during routine bacterial propagation.

1. Host Strains and Medium Configuration

• E. coli Propagation Strain (Critical): Never utilize conventional DH5a competent variants. Deploy engineered cell lines configured for large or unstable replicons, such as Stbl3 or SURE competent lineages.

• Cultivation Media parameters: Standard Lysogeny Broth (LB) liquid formulas or solid agar matrices supplemented with 100 ug/mL Ampicillin (or 50 ug/mL Kanamycin depending on backbone tags). To strictly mitigate recombination drift, execute growth steps at 30 degrees Celsius slow shaking rather than conventional 37 degrees Celsius runs.

2. Transformation and Revitalization Routine

1. Thaw an aliquot of 50–100 uL competent Stbl3 cells gently on a chilled ice bed.

2. Deliver 1–2 uL of the purified mouse Piezo1 plasmid DNA into the cell suspension. Mix via smooth flicking (do not vortex) and incubate on ice for 30 minutes.

3. Transfer the tube into a calibrated water bath set precisely at 42 degrees Celsius for a rigorous heat-shock window of 45 seconds. Instantly plunge the tube back into the ice bed for 2 minutes without agitation.

4. Inoculate the shocked cells with 500 uL of sterile, antibiotic-free LB broth or SOC recovery medium. Incubate horizontal in a shaking incubator at 30 degrees Celsius (recommended) running at 200 rpm for 60 minutes of out-growth recovery.

5. Concentrate the cells via quick centrifugation at 4,000 rpm for 3 minutes. Decant excess supernatant fluid, resuspend the pellet in the remaining ~100 uL volume, and spread evenly onto pre-warmed selective LB agar plates.

6. Incubate inverted at 30 degrees Celsius for 16–20 hours until distinct colonies materialize (growth kinetics are slowed at 30 degrees Celsius, requiring a longer wait than standard overnight runs).

3. Plasmid Proliferation and Endotoxin-Free Processing (Midiprep/Maxiprep)

1. Pick a singular, well-isolated colony from the selective agar plate and drop it into 5 mL of selective liquid LB broth. Pre-culture at 30 degrees Celsius running at 220 rpm for 6–8 hours.

2. Scale up the culture by inoculating a 1:500 volume split into 100–200 mL of selective liquid LB broth. Proliferate in a shaking incubator configured to 30 degrees Celsius running at 220 rpm for 14–16 hours (overnight).

3. Extraction Mandate: Because target assays directly quantify delicate cellular tension and calcium parameters, researchers must utilize Endotoxin-free Plasmid Extraction Midiprep/Maxiprep Kits. Residual bacterial lipopolysaccharides (LPS) cause cellular toxic shock, compromising mechanical baseline sensitivities and triggering non-specific calcium ion channels.

4. Elute the bound DNA matrix using sterile nuclease-free water. Verify purity via spectrophotometry and evaluate integrity by running 1 uL on a standard agarose gel to confirm a clean, non-degraded supercoiled macro-band matching the target mass profile.

4. Transient Mammalian Transfection Guide

Exemplified utilizing standard HEK293T or primary mouse endothelial lines seeded within 6-well plate platforms:

1. Target Confluency Baseline: Plate vegetative targets 24 hours prior to transfecting to lock in an optimal 60%–70% confluency density on the day of delivery.

2. Formulating Complex Master-Mixes (Per Single Well Parameters):

   • Tube A: Dilute 2.0–2.5 ug of ultrapure endotoxin-free mouse Piezo1 plasmid DNA within 100 uL of serum-free, protein-free Opti-MEM medium. Mix smoothly.

   • Tube B: Dilute 4–6 uL of dedicated liposome transfection reagents (e.g., Lipofectamine 2000 or 3000 systems) within 100 uL of serum-free Opti-MEM. Mix and let rest for 5 minutes.

3. Complexation Assembly: Blend the entire volume of Tube A directly into Tube B. Flick the bottom of the microfuge tube gently to mix and incubate undisturbed at room temperature for 15–20 minutes to secure stable plasmid-liposome lipoplex arrangements.

4. Inoculation Route: Aspirate spent culture medium from the well and replace with fresh low-serum or complete growth media. Supplement the layout by dripping the assembled lipoplex slurry uniformly across the target matrix. Gently rock the plate side-to-side.

5. Incubation & Downstream Monitoring: House the treated vessels at 37 degrees Celsius under a humidified 5% CO2 workspace. Refresh the well with premium complete culture media 4–6 hours post-transfection. Target mouse Piezo1 protein processing and cell surface expression maximize 24–48 hours post-transfection. At this window, administer Yoda1 chemical challenges, flow chamber shear stress profiling, or mechanical stretching vectors, tracking response profiles via Western Blotting, immunofluorescence confocal microscopy, or ratiometric live-cell calcium imaging loops.

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