BioVector® BJNhem20-TBXT-2A-EGFP 人胚胎干细胞报告株档案

一、 背景与来源说明

BJNhem20-TBXT-2A-EGFP 细胞株衍生自临床级人胚胎干细胞系（hESC line BJNhem20）。该报告株利用基因编辑技术（如 CRISPR/Cas9）在内源性 TBXT 基因（亦称 T 基因或 Brachyury 基因，人中胚层/原条的关键标志物）的 C 端融合插入了 2A-EGFP 表达盒。在干细胞多能性维持状态下（未诱导），细胞不表达 GFP；当向中胚层（Mesoderm）或原条（Primitive Streak）方向诱导分化时，内源 TBXT 启动子激活，表达 TBXT 蛋白的同时通过 2A 自剪切肽协同表达 EGFP 荧光，是研究早期胚胎发育、原条形成、脊索发育以及中胚层谱系分化的实时活细胞荧光报告模型。

二、 细胞生物学特征

• 组织来源：人（Homo sapiens）胚胎干细胞系（hESC line BJNhem20，源自内细胞团 ICM）

• 生长特性：贴壁生长，呈典型干细胞克隆（Colony）形态，克隆边缘清晰，核质比高

• 报告基因构建原理：

  • 靶基因：TBXT (T-box transcription factor T / Brachyury)

  • 工程化形式：Endogenous TBXT-2A-EGFP 融合作动表达（未破坏内源 TBXT 蛋白功能）

  • 荧光信号：在中胚层/原条诱导阶段发出绿色荧光（EGFP，激发光 ~488 nm，发射光 ~507 nm）

• 多能性标记：未分化状态下强表达 OCT4、SOX2、NANOG、SSEA-4、TRA-1-60 等 Stemness 标志物。

三、 培养条件与分化诱导指南

1. 未分化干细胞维持培养（Feeder-Free Culture）

• 推荐基质包被：Matrigel（1:100 稀释包被）或 Recombinant Human Vitronectin / Laminin-521

• 完全培养基配方：

  • 基础培养基：mTeSR1、mTeSR Plus 或 StemFlex 培养基

  • 消化/传代试剂：ReLeSR 或 0.5 mM EDTA（温和无酶克隆传代），避免使用 Trypsin 以防止干细胞异常分化

  • Small Molecule：初次接种及冻存复苏时添加 10 $\mu$M Y-27632（ROCK 抑制剂） 以提高细胞存活率

• 培养环境：37°C，5% $\text{CO}_2$，饱和湿度（推荐 5% $\text{O}_2$ 缺氧/生理氧培养环境，有利于保持多能性）

2. 中胚层（TBXT+）诱导与荧光观测

• 诱导条件：利用含 CHIR99021（GSK3$\beta$ 抑制剂，激活 Wnt 通路）及 BMP4 的中胚层分化培养基处理 24–48 小时。

• 荧光检测：诱导 24-36 小时后可在荧光显微镜下观察到克隆边缘或整个克隆启动表达强 EGFP 绿色荧光，可通过流式细胞术（FACS）按 GFP+ 比例定量分选提取早期中胚层前体细胞。

四、 技术指标简表

BioVector® BJNhem20-TBXT-2A-EGFP Human Embryonic Stem Cell Reporter Line Datasheet

1. Background & Context

The BJNhem20-TBXT-2A-EGFP reporter cell line was established from the human embryonic stem cell (hESC) line BJNhem20 using precise genome editing (e.g., CRISPR/Cas9). A 2A-EGFP reporter cassette was knocked into the 3' end of the endogenous TBXT gene (also known as T or Brachyury, a master transcriptional regulator of the primitive streak and nascent mesoderm). In pluripotency maintenance culture, GFP expression is silent. Upon mesodermal or primitive streak lineage specification, activation of the endogenous TBXT promoter triggers co-expression of TBXT and EGFP. This reporter provides a non-invasive, live-cell readout for studying gastrulation, early mesoderm specification, and lineage fate decisions.

2. Biological Characteristics

• Organism & Origin:Homo sapiens, human embryonic stem cell line (BJNhem20, derived from inner cell mass).

• Growth Properties: Adherent colonies with well-defined borders and high nuclear-to-cytoplasmic ratio under feeder-free conditions.

• Reporter Architecture:

  • Target Gene:TBXT (T-box transcription factor T / Brachyury).

  • Knock-in Strategy: Endogenous TBXT-2A-EGFP bicistronic locus (preserves endogenous TBXT protein function).

  • Fluorophore: Enhanced Green Fluorescent Protein (EGFP; Excitation ~488 nm, Emission ~507 nm).

• Pluripotency Profile: High expression of OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60 in the undifferentiated state.

3. Culture & Handling Protocols

3.1 Feeder-Free Maintenance Culture

• Substrate Coating: Matrigel (diluted 1:100) or Recombinant Human Vitronectin / Laminin-521.

• Culture Medium:

  • Complete Medium: mTeSR1, mTeSR Plus, or StemFlex medium.

  • Passaging Reagents: ReLeSR or 0.5 mM EDTA in PBS (gentle non-enzymatic clump passaging). Avoid single-cell trypsinization for routine maintenance.

  • Survival Supplement: Add 10 $\mu$M Y-27632 (ROCK inhibitor) during thawing and cell seeding to prevent anoikis.

• Incubation Parameters: 37°C, 5% $\text{CO}_2$, humidified incubator (5% $\text{O}_2$ physiological hypoxia recommended).

3.2 Mesodermal Differentiation & Reporter Readout

• Induction Protocol: Treat undifferentiated colonies with mesoderm differentiation medium supplemented with CHIR99021 (GSK3$\beta$ inhibitor / Wnt activator) and BMP4 for 24–48 hours.

• EGFP Detection: EGFP fluorescence becomes visually discernible via fluorescence microscopy within 24–36 hours post-induction. EGFP+ cell populations can be quantified and isolated via Fluorescence-Activated Cell Sorting (FACS).

4. Technical Data Summary

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