MDCK-SIAT1 BioVector® Cell Line犬肾脏上皮细胞株-流感疫苗开发生产用-BioVector NTCC典型培养物保藏中心

BioVector® MDCK-SIAT1 Cell Line

English Version

1. Cell Line Overview

• Cell Line Name: BioVector® MDCK-SIAT1

• Accession Number: CVCL_Z936

• RRID: CVCL_Z936

• Species of Origin:Canis familiaris (Dog), Cocker Spaniel breed

• Tissue Origin: Kidney

• Cell Type: Epithelial

• Morphology: Adherent

• Parental Line: BioVector® MDCK (Madin-Darby Canine Kidney)

• Derivation: Stable transfection of MDCK cells with cDNA encoding human α-2,6-sialyltransferase (SIAT1) in a bicistronic expression vector

• ECACC Number: 05071502

• BEI Resources Number: NR-60144

• Depositor: Dr. Mikhail Matrosovich, Institute of Virology, Philipps University, Marburg, Germany

2. Description and Applications
BioVector® MDCK-SIAT1 is a genetically modified variant of the standard MDCK cell line, engineered to overexpress the human α-2,6-sialyltransferase (SIAT1) enzyme. This enzyme adds α-2,6-linked sialic acids to glycoproteins and glycolipids, which serve as the primary receptors for human influenza viruses.

The modification results in cells expressing twofold higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids compared to parental MDCK cells. This makes MDCK-SIAT1 cells more closely resemble human airway epithelial cells, providing a more physiologically relevant model for studying human influenza viruses.

These cells are particularly valuable for testing the sensitivity of human influenza viruses to neuraminidase inhibitors (NAIs) such as oseltamivir, as they show increased sensitivity to these antiviral compounds due to the enhanced expression of human-type receptors.

3. Key Features

• Enhanced Human-Type Receptor Expression: Overexpresses α-2,6-linked sialic acids (human influenza virus receptors)

• Reduced Avian-Type Receptor Expression: Lower expression of α-2,3-linked sialic acids (avian influenza virus receptors)

• NAI Sensitivity Testing: Ideal system for evaluating influenza virus susceptibility to neuraminidase inhibitors

• Clinical Isolate Propagation: Improved binding and propagation of clinical human influenza virus isolates

• GMO Status: Genetically Modified Organism Class 1 (GMO1)

4. Growth and Culture Conditions

• Recommended Medium: BioVector® High-Glucose DMEM (4.5 g/L glucose) with 4 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate, supplemented with:

  • 10% fetal bovine serum (FBS)

  • 1 mg/mL Geneticin (G418) for selection pressure

• Growth Characteristics: Adherent epithelial cells; doubling time approximately 21-31 hours

• Culture Conditions: 37°C, 5% CO₂

• Subculture Routine:

  • Split sub-confluent cultures (85-95%) at ratios of 1:10 to 1:20

  • Seeding density: 2-4 × 10⁴ cells/cm²

  • Use 0.05% trypsin-EDTA for dissociation (3-15 minutes at 37°C)

  • Centrifuge at 125-200 × g for 8-10 minutes during passaging

• Medium Renewal: Every 2-3 days

5. Quality Control and Safety

• Sterility Testing: Negative for bacteria, fungi, and mycoplasma

• Biosafety Level: BSL-1; ACDP Hazard Group 2

• GMO Classification: Genetically Modified Organism Class 1 (GMO1)

6. Storage and Handling

• Storage Conditions: Store at -100°C or colder, preferably in vapor phase of liquid nitrogen. Storage at -70°C will result in loss of viability.

• Thawing Procedure: Rapidly thaw in 37°C water bath with gentle agitation; centrifuge to remove cryoprotectant; resuspend in pre-warmed growth medium.

7. Applications

• Antiviral Drug Testing: Evaluation of influenza virus sensitivity to neuraminidase inhibitors (oseltamivir, zanamivir)

• Influenza Virus Isolation: Propagation of human influenza A and B viruses from clinical specimens

• Virus-Host Interaction Studies: Investigation of receptor specificity and viral binding mechanisms

• Drug Resistance Monitoring: Detection of neuraminidase inhibitor-resistant influenza strains

• Vaccine Development: Production of influenza virus isolates for vaccine candidate development

BioVector® MDCK-SIAT1 细胞系说明书

中文版本

1. 细胞系概述

• 细胞系名称： BioVector® MDCK-SIAT1

• 登录号： CVCL_Z936

• RRID： CVCL_Z936

• 物种来源： 犬，可卡犬品种

• 组织来源： 肾脏

• 细胞类型： 上皮细胞

• 形态特征： 贴壁生长

• 亲本系： BioVector® MDCK（马-达二氏犬肾细胞）

• 衍生方法： 通过双顺反子表达载体将人α-2,6-唾液酸转移酶（SIAT1）的cDNA稳定转染至MDCK细胞中获得

• ECACC编号： 05071502

• BEI Resources编号： NR-60144

• 保藏者： Dr. Mikhail Matrosovich, Institute of Virology, Philipps University, Marburg, Germany

2. 描述与应用
BioVector® MDCK-SIAT1是标准MDCK细胞系的基因修饰变体，经工程化改造以过表达人α-2,6-唾液酸转移酶（SIAT1）。该酶负责在糖蛋白和糖脂上添加α-2,6连接的唾液酸，这些唾液酸是人流感病毒的主要受体。

该修饰使细胞与亲本MDCK细胞相比，表达两倍量的6-连接唾液酸和一半量的3-连接唾液酸。这使得MDCK-SIAT1细胞更接近于人气道上皮细胞，为研究人流感病毒提供了更贴近生理的模型。

这些细胞特别适用于测试人流感病毒对神经氨酸酶抑制剂（NAIs）如奥司他韦的敏感性，由于人型受体表达增强，它们对这些抗病毒化合物表现出更高的敏感性。

3. 主要特征

• 增强的人型受体表达： 过表达α-2,6-连接唾液酸（人流感病毒受体）

• 降低的禽型受体表达： α-2,3-连接唾液酸（禽流感病毒受体）表达降低

• NAI敏感性测试： 评估流感病毒对神经氨酸酶抑制剂敏感性的理想系统

• 临床分离株扩增： 改善人流感病毒临床分离株的结合与扩增

• GMO状态： 1类基因修饰生物体（GMO1）

4. 生长与培养条件

• 推荐培养基： BioVector® 高糖DMEM（4.5 g/L葡萄糖），含4 mM L-谷氨酰胺、1 mM丙酮酸钠和1.5 g/L碳酸氢钠，添加：

  • 10%胎牛血清（FBS）

  • 1 mg/mL Geneticin（G418）用于选择压力

• 生长特性： 贴壁上皮细胞；倍增时间约21-31小时

• 培养条件： 37°C，5% CO₂

• 传代常规：

  • 亚融合状态（85-95%）下按1:10至1:20比例传代

  • 接种密度：2-4 × 10⁴ 细胞/cm²

  • 使用0.05%胰蛋白酶-EDTA消化（37°C下3-15分钟）

  • 传代过程中125-200 × g离心8-10分钟

• 培养基更换： 每2-3天

5. 质量控制与安全性

• 无菌检测： 细菌、真菌和支原体检测均为阴性

• 生物安全等级： BSL-1；ACDP危险组2级

• GMO分类： 1类基因修饰生物体（GMO1）

6. 储存与处理

• 储存条件： 储存于-100°C或更低温度，优选液氮气相中。在-70°C储存会导致活力丧失。

• 复苏步骤： 在37°C水浴中快速解冻，轻轻搅动；离心去除冷冻保护剂；重悬于预温的生长培养基中。

7. 应用

• 抗病毒药物测试： 评估流感病毒对神经氨酸酶抑制剂（奥司他韦、扎那米韦）的敏感性

• 流感病毒分离： 从临床样本中扩增人甲型和乙型流感病毒

• 病毒-宿主相互作用研究： 研究受体特异性和病毒结合机制

• 耐药性监测： 检测神经氨酸酶抑制剂耐药的流感病毒株

• 疫苗开发： 为候选疫苗开发生产流感病毒分离株

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