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mRNA / cRNA 注射及卵母细胞蛋白表达mRNA/cRNA injection & oocyte protein expression

向爪蟾卵母细胞显微注射 mRNA / cRNA，实现离子通道、转运蛋白、受体及膜蛋白的异源表达；配合 双电极电压钳（TEVC）功能检测、FLAG / GFP 标签融合表达、Western Blot 蛋白定量及激光共聚焦显微镜膜定位验证，提供从分子到功能的全面数据包。Microinjection of mRNA/cRNA into Xenopus oocytes for heterologous expression of ion channels, transporters, receptors and membrane proteins — combined with TEVC functional recording, FLAG/GFP-tag fusion expression, Western Blot quantification and confocal membrane-localization imaging for a complete molecular-to-function data package.

cRNA 制备、细胞质注射、恢复培养与膜蛋白表达的流程。

为什么用爪蟾卵母细胞？Why Xenopus oocytes?

爪蟾卵母细胞体积大、内源通道背景低、翻译与翻译后修饰机制完善，是异源表达膜蛋白并进行电生理 / 转运功能研究的经典系统。Large, with low endogenous channel background and full translational machinery, Xenopus oocytes are the classic system for heterologous membrane-protein expression and electrophysiology.

  • 离子通道：钾/钠/钙/阴离子通道电流记录与药理学Ion channels: K/Na/Ca/anion currents & pharmacology
  • 转运蛋白：HKT、MATE、ABC、NRT、MTP 等转运活性测定Transporters: HKT/MATE/ABC/NRT/MTP transport assays
  • 受体：NMDA 受体、嗅觉/信息素受体功能鉴定Receptors: NMDA, olfactory/pheromone receptors
  • 水通道：PIP 等 aquaporin 通透性与定位Aquaporins: PIP permeability & localization
  • 膜蛋白验证：FLAG/GFP 标签融合，Western Blot 定量，共聚焦确认质膜定位Membrane protein verification: FLAG/GFP fusion, Western Blot quantification, confocal plasma-membrane localization
膜蛋白表达验证Membrane protein verification

FLAG / GFP 标签 · Western Blot · 激光共聚焦显微镜FLAG/GFP tag · Western Blot · Confocal microscopy

在完成电生理或转运功能检测的同时，中心提供分子标签融合表达与多模态验证服务，从蛋白表达量和亚细胞定位两个维度确认目的蛋白正确插入质膜，为功能数据提供分子层面佐证。Alongside electrophysiology or transport assays, we provide molecular-tag fusion expression with multi-modal verification — quantifying protein expression and confirming plasma-membrane localization to support your functional data at the molecular level.

01 · 标签构建Tag construction

FLAG / GFP / HA / mCherry 融合表达FLAG / GFP / HA / mCherry fusion expression

在目的蛋白 N 端或 C 端插入标签序列（客户自建或中心协助克隆），体外转录获得带标签 cRNA，注射卵母细胞后 1–3 天检测。GFP / mCherry 可直接荧光成像；FLAG / HA 可用于免疫染色、Co-IP 等。Tag sequences are inserted at the N- or C-terminus (client-built or center-cloned), transcribed to tagged cRNA and injected. GFP/mCherry enable direct live-cell imaging; FLAG/HA enable immunostaining and Co-IP.

02 · Western Blot

蛋白表达量与修饰状态检测Protein expression level & modification status

注射后按时间点收集卵母细胞，裂解提取总蛋白或质膜蛋白组分，SDS-PAGE 分离后用抗 FLAG / GFP / HA 抗体或目的蛋白特异抗体（客户提供）进行 Western Blot 检测。评估表达量、分子量及糖基化等翻译后修饰，以水注射对照组排除内源蛋白干扰。Oocytes are harvested at defined time-points; total or plasma-membrane protein fractions are resolved by SDS-PAGE and detected with anti-FLAG/GFP/HA antibodies or client-supplied target-specific antibodies. This evaluates expression level, molecular weight and glycosylation, compared against water-injected controls.

03 · 共聚焦成像Confocal imaging

质膜定位实时可视化Real-time plasma-membrane localization

对注射了 GFP / mCherry 融合 cRNA 的卵母细胞进行激光共聚焦成像，活细胞状态下直接观察蛋白亚细胞定位。正确定位于质膜的蛋白呈现清晰的质膜环状荧光；内质网或内体滞留则提示需优化构建体。可配合 DiI / DiO 膜染料或凝集素（Lectin）进行共定位分析，确认质膜归属。Live-cell laser confocal imaging of oocytes expressing GFP/mCherry-fused proteins directly visualizes subcellular localization. Correct plasma-membrane targeting appears as a distinct fluorescent ring at the cell periphery; ER or endosome retention indicates construct optimization is needed. Co-localization with DiI/DiO dyes or lectin confirms plasma-membrane identity.

验证方法Method检测指标Readout所需标签 / 抗体Required tag / antibody交付物Deliverables
Western Blot蛋白分子量与表达量MW & expression levelFLAG / GFP / HA（或客户自备抗体）凝胶图 + 定量图表Gel image + quantification
激光共聚焦Confocal质膜 / 细胞器定位PM / organelle localizationGFP / mCherry / YFP高分辨率荧光图像 + Z 轴扫描High-res images + Z-stack
质膜蛋白分级Membrane fractionation质膜蛋白富集确认PM enrichment任意标签或自备抗体Any tag or custom antibody分级 WB + 比较数据Fractionation WB + comparison
服务流程Workflow

从 cRNA 到多维度数据From cRNA to multi-dimensional data

1
基因克隆与 cRNA 体外转录（可含 FLAG / GFP 标签）Gene cloning & cRNA synthesis (with optional FLAG/GFP)
2
卵母细胞显微注射Oocyte microinjection
3
表达孵育（1–4 天）Expression incubation (1–4 d)
4
表达验证（Western Blot / 共聚焦定位）Expression verification (Western / confocal)
5
TEVC 电生理 / 转运 / 渗透率功能检测与分析TEVC / transport / permeability assay & analysis
代表性成果Selected publications

客户使用本服务发表的论文（节选）Client papers using this service

点击 DOI 链接可在新窗口查看原文。完整名单见 客户 SCI 论文精选。Click the DOI link to view each paper. Full list: client SCI papers.

Molecular Plant2026
An SAPK10-RISBZ1-ABT axis promotes endosperm-to-embryo ABA transport to enhance pre-harvest sprouting tolerance in rice
Li et al. · Mol Plant · 在线发表online ahead of print · ABA 外排转运蛋白 ABT（ABC 家族）ABA efflux transporter ABT (ABC family)
DOI
PNAS2026
A BRI1-CNGC12 phosphorylation module links hormone signaling to manganese homeostasis in plants
Zhang et al. · PNAS 123(10):e2514483123 · CNGC12 钙通道 / 锰稳态CNGC12 Ca²⁺ channel / Mn homeostasis
DOI
Nature Communications2025
CPK1 activates CNGCs through phosphorylation for Ca²⁺ signaling to promote root hair growth in Arabidopsis
Zhu et al. · Nat Commun 16:676 · 钙信号通道 CNGCCNGC Ca²⁺ channel
DOI
Cell Reports2025
Regulation of the non-selective Na⁺ importer capacity of NRT1.2/NPF4.6/AIT1 by SOS2-mediated phosphorylation in Arabidopsis
Liu et al. · Cell Rep 44(6):115729 · 钠转运蛋白 NRT1.2Na⁺ transporter NRT1.2
DOI
Plant Cell & Environment2025
Ethylene-mediated RsCBF2 and RsERF18 enhance salt tolerance by directly regulating aquaporin gene RsPIP2-1 in radish
Yi et al. · Plant Cell Environ 48(8):5740–5752 · 水通道 RsPIP2-1 / 耐盐Aquaporin RsPIP2-1 / salt tolerance
DOI
J Integr Plant Biol2025
The ABC transporter SmABCG1 mediates tanshinones export from the peridermic cells of Salvia miltiorrhiza root
Li et al. · J Integr Plant Biol 67(1):135–149 · ABC 转运蛋白ABC transporter
DOI
Plant Communications2024
Two aquaporins, PIP1;1 and PIP2;1, mediate the uptake of neonicotinoid pesticides in plants
Wan et al. · Plant Commun 5(5):100830 · 水通道 / 农药吸收Aquaporins / pesticide uptake
DOI
Nature2021
Structural basis of ketamine action on human NMDA receptors
Zhang et al. · Nature 596(7871):301–305 · NMDA 受体NMDA receptor
DOI
Neuron2021
Gating mechanism and a modulatory niche of human GluN1-GluN2A NMDA receptors
Wang et al. · Neuron 109(15):2443–2456 · NMDA 受体门控NMDA gating
DOI
J Integr Plant Biol2023
Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon
Liu et al. · J Integr Plant Biol 65(10):2304–2319 · MATE 转运蛋白MATE transporter
DOI
Molecular Plant2022
Ca²⁺-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis
Ju et al. · Mol Plant 15(3):419–437 · 液泡锰转运蛋白 MTP8Vacuolar Mn transporter MTP8
DOI
Cell Calcium2021
The expression of constitutively active CPK3 impairs potassium uptake and transport in Arabidopsis under low K⁺ stress
Ren et al. · Cell Calcium 98:102447 · 钾吸收 / 钙依赖蛋白激酶K⁺ uptake / CPK3
DOI
J Plant Physiol2021
Slow anion channel GhSLAC1 is essential for stomatal closure in response to drought stress in cotton
Ren et al. · J Plant Physiol 258–259:153360 · 阴离子通道Anion channel
DOI
iScience2021
A single point mutation causes one-way alteration of pheromone receptor function in two Heliothis species
Cao et al. · iScience 24(9):102981 · 信息素受体Pheromone receptor
DOI
Front Plant Sci2020
Transmembrane helices 2 and 3 determine the localization of plasma membrane intrinsic proteins in eukaryotic cells
Wang et al. · Front Plant Sci 10:1671 · PIP 水通道定位PIP aquaporin localization
DOI
Br J Pharmacol2020
Cloning and characterization of the rat Slo3 (KCa5.1) channel: from biophysics to pharmacology
Wang et al. · Br J Pharmacol 177(15):3552–3567 · 钙激活钾通道Ca²⁺-activated K⁺ channel
DOI
Plant Mol Biol2020
Supportive role of the Na⁺ transporter CmHKT1;1 from Cucumis melo in transgenic Arabidopsis salt tolerance through improved K⁺/Na⁺ balance
Gao et al. · Plant Mol Biol 103(4-5):561–580 · HKT 钠转运蛋白HKT Na⁺ transporter
DOI

表达你的目标蛋白Express your protein of interest

提供基因序列与实验目标，我们完成 cRNA 合成、注射与 TEVC 检测。Send your gene and goal — we handle cRNA, injection and TEVC.

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