BioVector? Mouse Piezo1 Overexpression Plasmid / 鼠源 Piezo1 過表達(dá)質(zhì)粒

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 質(zhì)粒名稱：鼠源 Piezo1 過表達(dá)質(zhì)粒（Mouse Piezo1 Overexpression Plasmid）。

• 目的基因背景（Piezo1）：

  • 基因別名：Piezo1, Fam38a。

  • 物種來源：小鼠（Mus musculus）。

  • 蛋白結(jié)構(gòu)與功能：Piezo1 是一種進(jìn)化上高度保守的由機(jī)械力激活的非選擇性陽離子通道（Mechanosensitive Ion Channel）。小鼠 Piezo1 單體極其龐大，由多達(dá) 2547 個(gè)氨基酸殘基組成，包含多達(dá) 38 個(gè)跨膜螺旋區(qū)域。在天然狀態(tài)下，三個(gè) Piezo1 單體組裝成一個(gè)獨(dú)特的、形似“三葉螺旋槳”的同源三聚體結(jié)構(gòu)，在細(xì)胞膜表面形成一個(gè)向內(nèi)凹陷的巨大穹窿或“納米圓頂”（Nano-dome）。

  • 通透性特性：當(dāng)細(xì)胞膜受到機(jī)械刺激（如流體剪切力、滲透壓改變、牽張拉伸或牽引力）時(shí)，Piezo1 通道會迅速開放，主要介導(dǎo) $Ca^{2+}$（鈣離子）的快速內(nèi)流，同時(shí)通透 $Na^+$、$K^+$ 等單價(jià)陽離子。這能將物理機(jī)械信號瞬時(shí)轉(zhuǎn)譯為細(xì)胞內(nèi)的生物化學(xué)信號（如激活鈣信號通路、NF-AT、FAK 等下游級聯(lián)）。

• 載體構(gòu)件特征：

  • 強(qiáng)啟動子：目的基因通?？寺∮谟?CMV 或 EF1a 強(qiáng)啟動子驅(qū)動的哺乳動物高效表達(dá)載體（如 pCDNA3.1(+)、pEGFP-N1 或 pLenti 慢病毒載體）中，以確保在宿主細(xì)胞內(nèi)實(shí)現(xiàn)高水平的結(jié)構(gòu)性過表達(dá)。

  • 標(biāo)簽/熒光蛋白融合（依據(jù)具體批次變體）：可選融合 FLAG、HA、Myc 等小肽標(biāo)簽，或在 C 端/N 端融合 GFP/mCherry 熒光蛋白，便于后期通過 Western Blot（WB）、免疫共沉淀（Co-IP）或活細(xì)胞熒光成像追蹤 Piezo1 在細(xì)胞膜上的空間定位。

• 抗性篩選標(biāo)記：

  • 大腸桿菌抗性：氨芐青霉素抗性（Ampicillin, AmpR） 或 卡那霉素抗性（Kanamycin, KanR），用于工程菌擴(kuò)增。

  • 哺乳動物細(xì)胞篩選抗性：通常帶有 新霉素（G418/Neomycin）、嘌呤霉素（Puromycin）或潮霉素（Hygromycin B）抗性標(biāo)記。

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

鼠源 Piezo1 過表達(dá)質(zhì)粒是開展生物力學(xué)（Mechanobiology）和血管/骨骼發(fā)育等前沿領(lǐng)域研究的基石工具：

1. 細(xì)胞機(jī)械感應(yīng)與物理力轉(zhuǎn)導(dǎo)研究（Mechanotransduction）：

   用于深入探究細(xì)胞如何感知周圍微環(huán)境的硬度或外界壓力。過表達(dá) Piezo1 后，細(xì)胞對流體剪切力（Fluid Shear Stress）或細(xì)胞劃痕拉伸的敏感度大幅上調(diào)。常用于研究血管內(nèi)皮細(xì)胞在血流剪切力下的重塑、成骨細(xì)胞在重力/機(jī)械載荷下的骨形成，以及免疫細(xì)胞（如巨噬細(xì)胞）在硬質(zhì)基質(zhì)中的激活機(jī)制。

2. 細(xì)胞鈣信號動力學(xué)監(jiān)測（Calcium Signaling Kinetics）：

   通過將該質(zhì)粒轉(zhuǎn)染至易于操作的工具細(xì)胞（如 HEK293T）中，配合 Fluo-4 AM 等鈣離子熒光探針或高敏感鈣成像系統(tǒng)，可體外重現(xiàn)由物理壓迫或 Piezo1 特異性小分子激動劑（如 Yoda1, Jedi1/2）誘導(dǎo)的特征性鈣內(nèi)流脈沖，用于驗(yàn)證該通道的電生理活性。

3. 組織器官發(fā)育與病理機(jī)制建模：

   小鼠 Piezo1 在紅細(xì)胞體積調(diào)節(jié)、血管新生、淋巴管發(fā)育以及上皮細(xì)胞穩(wěn)態(tài)維持中扮演關(guān)鍵角色。利用該質(zhì)粒在特定原代細(xì)胞中實(shí)施過表達(dá)，可用于模擬和研究剪切力敏感性遺傳性干癟紅細(xì)胞增多癥（Hereditary Xerocytosis）、高血壓導(dǎo)致的血管壁肥厚、以及腫瘤微環(huán)境硬度改變引發(fā)的癌細(xì)胞遷移和侵襲。

三 實(shí)驗(yàn)室質(zhì)粒轉(zhuǎn)化、擴(kuò)增、高純度提取與轉(zhuǎn)染標(biāo)準(zhǔn)步驟

1. 擴(kuò)增菌株與培養(yǎng)基配置

由于 Piezo1 的編碼序列（CDS）長達(dá) 7.6 kb 左右，整包質(zhì)粒通常超過 12-14 kb，屬于巨大的大質(zhì)粒，普通質(zhì)粒常規(guī)擴(kuò)增極易發(fā)生自發(fā)性同源重組導(dǎo)致目的片段缺失。

• 推薦大腸桿菌宿主：嚴(yán)禁使用常規(guī) DH5a 擴(kuò)增。強(qiáng)烈推薦選用專為大質(zhì)粒、不穩(wěn)定性重復(fù)序列設(shè)計(jì)的 Stbl3 或 SURE 感受態(tài)細(xì)胞。

• 培養(yǎng)基與抗性配置：LB 肉湯/固體瓊脂，添加最終工作濃度為 100 ug/mL 氨芐青霉素（Ampicillin，或依說明書改用 50 ug/mL 卡那霉素）。為了防止大質(zhì)粒重組，建議將搖菌擴(kuò)增溫度由常規(guī)的 37 攝氏度降低至 30 攝氏度過夜慢搖。

2. 大腸桿菌轉(zhuǎn)化與復(fù)蘇步驟

1. 取出 50 - 100 uL 的大腸桿菌 Stbl3 感受態(tài)細(xì)胞置于冰上緩慢融化。

2. 加入 1 - 2 uL 鼠源 Piezo1 過表達(dá)質(zhì)粒 DNA，輕彈管底混勻（嚴(yán)禁渦旋震蕩），冰浴 30 分鐘。

3. 將離心管迅速置于 42 攝氏度水浴中精確熱擊 45 秒，隨后立即插回冰中靜置 2 分鐘，切勿搖動。

4. 向管內(nèi)加入 500 uL 不含抗生素的無菌 LB 肉湯（或 SOC 培養(yǎng)基），置于 30 攝氏度（或 37 攝氏度）振蕩培養(yǎng)箱內(nèi)，以 200 rpm 復(fù)蘇勻速搖菌 60 分鐘。

5. 4000 rpm 離心 3 分鐘，棄去部分上清，留約 100 uL 液體將菌體吹勻，平鋪涂布于含有對應(yīng)抗性的 LB 固體平板上。

6. 置于 30 攝氏度（建議）培養(yǎng)箱中倒置培養(yǎng) 16 - 20 小時(shí)（若在 30 攝氏度生長較慢，可能需要更長時(shí)間，直至長出清晰可見的健壯單菌落）。

3. 質(zhì)粒擴(kuò)增與無內(nèi)毒素提?。∕idiprep / Maxiprep）

1. 挑取平板上邊緣清晰的單個(gè)單菌落，接種至 5 mL 含抗生素的 LB 液體肉湯試管中，30 攝氏度、220 rpm 預(yù)培養(yǎng) 6 - 8 小時(shí)。

2. 隨后按 1:500 比例擴(kuò)增接種至 100 - 200 mL 含抗生素的液體 LB 培養(yǎng)基中，置于 30 攝氏度 連續(xù)振蕩過夜培養(yǎng) 14 - 16 小時(shí)。

3. 提取關(guān)鍵：由于過表達(dá)質(zhì)粒后續(xù)直接用于哺乳動物細(xì)胞轉(zhuǎn)染，且 Piezo1 對細(xì)胞狀態(tài)極為敏感，必須使用無內(nèi)毒素的質(zhì)粒中提/大提試劑盒（Endotoxin-free Plasmid Kit）。殘留的內(nèi)毒素會嚴(yán)重干擾細(xì)胞對機(jī)械力的感應(yīng)并引發(fā)非特異性鈣內(nèi)流。

4. 提取出的質(zhì)粒溶解于無核酸酶的滅菌水中，利用分光光度計(jì)測量濃度。對于超大質(zhì)粒，建議抽樣 1 uL 進(jìn)行常規(guī)瓊脂糖凝膠電泳（或測序鑒定），確保帶型單一、無降解、無條帶缺失。

4. 哺乳動物細(xì)胞轉(zhuǎn)染（Transient Transfection）

以常規(guī) HEK293T 或小鼠原代內(nèi)皮細(xì)胞在 T25 瓶（或 6 孔板）中的操作為例：

1. 細(xì)胞密度：轉(zhuǎn)染前一天接種細(xì)胞，確保在轉(zhuǎn)染當(dāng)天細(xì)胞密度達(dá)到 60% - 70% 融合度。

2. 轉(zhuǎn)染體系（以 6 孔板單孔為例）：

   • A管：將 2 - 2.5 ug 的高質(zhì)量純化小鼠 Piezo1 質(zhì)粒 DNA 稀釋于 100 uL 無血清的 Opti-MEM 培養(yǎng)基中，輕柔混勻。

   • B管：將 4 - 6 uL 脂質(zhì)體轉(zhuǎn)染試劑（如 Lipofectamine 2000 或 3000）稀釋于 100 uL 無血清的 Opti-MEM 中，混勻并靜置 5 分鐘。

3. 復(fù)合物組裝：將 A 管液體全部滴入 B 管中，輕彈混勻，在室溫下靜置 15 - 20 分鐘以形成穩(wěn)定的質(zhì)粒-脂質(zhì)體復(fù)合物。

4. 侵染：吸除細(xì)胞舊培養(yǎng)基，更換為新鮮的低血清或完全培養(yǎng)基（根據(jù)轉(zhuǎn)染試劑說明）。將復(fù)合物均勻滴加至孔內(nèi)，輕微前后晃動。

5. 維持與檢測：置于 37 攝氏度、5% CO2 孵箱中培養(yǎng) 4 - 6 小時(shí)后更換為新鮮的完全培養(yǎng)基。轉(zhuǎn)染 24 - 48 小時(shí)后，Piezo1 蛋白在細(xì)胞膜表面達(dá)到表達(dá)峰值。此時(shí)可施加 Yoda1 激動劑、流體剪切力或劃痕刺激，通過 Western Blot、免疫熒光或活細(xì)胞鈣成像（Calcium Imaging）檢測其功能性過表達(dá)指標(biāo)。

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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