Stemgent致力于通过提供由一些世界DJ干细胞科研人员所开发的专有试剂盒工具促进干细胞科学的发展,Stemgent的产品包括:针对多能性、自我更新和分化的小分子;mRNA转录因子;基质;细胞系;细胞因子;抗体等。
The expression of the four human transcription factors Oct4, Klf4, Sox2, and c-Myc has been shown to reprogram human fibroblasts into an embryonic stem (ES) cell-like state known as induced pluripotent stem (iPS) cells.1 The Stemgent VSVg Retrovirus Reprogramming Set: Human OKSM consists of four retroviruses pseudotyped with the Vesicular Stomatitis Virus G-protein (VSVg). Each amphotrophic vector is capable of expressing one of the four reprogramming factors when transduced into a range of mammalian cells. The VSVg Retrovirus Reprogramming Set: Human OKSM is functionally validated to reprogram human fibroblasts and is sufficient to productively reprogram two human fibroblast samples in a 6-well format. The VSVg Retrovirus Green Fluorescent Protein (GFP) is included as a transduction control. The expression of the four human transcription factors Oct4, Klf4, Sox2, and c-Myc has been shown to reprogram human fibroblasts into an embryonic stem (ES) cell-like state known as induced pluripotent stem (iPS) cells.1 The Stemgent VSVg Retrovirus Reprogramming Set: Human OKSM consists of four retroviruses pseudotyped with the Vesicular Stomatitis Virus G-protein (VSVg). Each amphotrophic vector is capable of expressing one of these four factors when transduced into a range of mammalian cells. The VSVg Retrovirus Reprogramming Set: Human OKSM is functionally validated to reprogram human fibroblasts and is sufficient to productively reprogram at least 2 human fibroblast samples in a 6-well format. The VSVg Retrovirus Green Fluorescent Protein (GFP) is included as a transduction control.
Product Specifications
Size
1 Set
Set Contents
- 1 vial - Stemgent VSVg Retrovirus hOct4 (Cat. No. 07-0055), 100 µl
- 1 vial - Stemgent VSVg Retrovirus hKlf4 (Cat. No. 07-0056), 100 µl
- 1 vial - Stemgent VSVg Retrovirus hSox2 (Cat. No. 07-0057), 100 µl
- 1 vial - Stemgent VSVg Retrovirus hc-Myc (Cat. No. 07-0058), 100 µl
- 1 vial - Stemgent VSVg Retrovirus GFP (Cat. No. 07-0059), 100 µl
Storage and Stability
Store at -70°C for up to 3 months. Keep frozen until use. Avoid freeze and thaw cycles.
Quality Control
The transduction titer was determined by viral RNA quantification and normalized by a co-efficiency method to the VSVg Retrovirus GFP. Transcription factor protein expression was verified by immunocytochemistry analysis of transduced human BJ fibroblasts. Reprogramming was confirmed by the generation of iPS cell colonies from human BJ fibroblasts, and the resuting iPS cell colonies were characterized for a panel of pluripotency markers by immunocytochemistry. Each factor has been tested and found to be negative for Mycoplasma sp. by PCR and microbial contamination by a sterility test.
产品特点
1.快速 Fast
Stemgent® MicroRNA-Enhanced mRNA Reprogramming System只需2周即可将靶细胞重编程为iPS细胞,与仙台病毒(Sendai Virus)、非整合型DNA载体(Episomal DNA Vectors)等传统iPS重编程方法相比,缩短实验周期超过50%以上(Figure. 1)。
Figure 1. Stemgent® mRNA重编程系统、仙台病毒依赖的重编程系统以及游离型DNA载体依赖的重编程系统重编程时程比较。
简便 Easy与病毒、DNA载体依赖的iPS重编程手段不同,使用Stemgent® MicroRNA-Enhanced mRNA Reprogramming System对靶细胞进行重编程,生成的iPS细胞无需进行复杂耗时的下游筛选程序,只需简简单单6个步骤,即可轻松构建iPS细胞系。
| Description | Day |
Step 1 | Material Preparation | Prior to starting |
Step 2 | Plate Cells | 0 |
Step 3 | Transfect Cells | 1 to 12 |
Step 4 | Identify Cells | 13-14 |
Step 5 | Pick and Passage iPS Cell Colonies | 15-16 |
Step 6 | Maintain iPS Cell Cultures | 16+ |
注:因所用细胞类型及实验条件不同,上述时间轴可能略有不同,仅供参考。
Figure 2. Stemgent® MicroRNA-Enhanced mRNA Reprogramming System重编程过程时间表、显微镜下观察到的重编程过程中靶细胞形态变化。第12天,形成的iPS克隆表达多能型Marker TRA-1-81。
GX Efficient病毒、DNA载体等依赖的iPS重编程技术,其重编程效率介于0.00001%到1%之间不等,而Stemgent® MicroRNA-Enhanced mRNA Reprogramming System的重编程效率远远大于1%,极大地提高了iPS克隆的产出率。
表1. 不同类型iPS重编程手段效率比较
Reprogramming Method | Efficiency | Integrating | Screening |
RNA | >1% | No | No |
仙台病毒(Sendai virus) | 0.01-1% | No | Yes |
DNA载体(Episomal/Minicircle) | 0.00% | Possible | Yes |
慢病毒(Lentivirus) | 0.001-0.01% | Yes | Yes |
腺病毒(Adenovirus) | 0.0001-0.001% | Possible | Yes |
Protein | 0.00% | No | No |
某些iPS研究领域,例如iPS临床研究、ZL应用等,对iPS细胞的安全性提出了极高的要求。使用Stemgent® MicroRNA-Enhanced mRNA Reprogramming System重编程靶细胞,无需担心构建的iPS细胞系可能含有病毒载体残留,亦无需担心无关的基因整合进宿主基因组,是目前适用于临床研究及ZL的最理想选择。安全 Safe而以仙台病毒(Sendai Virus)为载体的iPS重编程手段,其病毒基因组虽然不会整合进宿主染色体,仙台病毒本身也不具备复制能力,但是为了构建真正意义上的virus-free的iPS细胞系,通常需要经过一个10-20代、耗时费力的筛选过程,以确保ZZ得到的iPS细胞系无病毒载体残留。以慢病毒(Lentivirus)体的iPS重编程手段,病毒基因组能够整合进宿主基因组,会给后续的iPS细胞分析带来干扰。
高性价比 Cost Effective 以仙台病毒为载体的iPS重编程试剂盒非常昂贵,综合重编程效率、时间及劳动力成本、安全性等多重考虑,Stemgent® MicroRNA-Enhanced mRNA Reprogramming System无疑是性价比的上乘之选。