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翻译:ReproRNA™-OKSGM

用于生成iPS细胞的非整合、自我复制的RNA重编程载体
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产品号 #05930_C

用于生成iPS细胞的非整合、自我复制的RNA重编程载体

产品优势

  • 非病毒，非积分载体系统
  • 自我复制载体只需要一次转染
  • 向量包含所有重编程因素
  • 成纤维细胞重编程效率与仙台病毒相当

产品组分包括

  • ReproRNA™-OKSGM试剂盒（目录#05930）
    • ReproRNA™-OKSGM载体（目录#05931）
    • ReproRNA™转染试剂
    • ReproRNA™转染补充
    • 重组B18R蛋白（目录#78075）
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Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.
  1. Trypsin-EDTA (0.25%)
    Trypsin-EDTA (0.25%)

    Enzymatic cell dissociation reagent

  2. DMEM with 1000 mg/L D-Glucose
    DMEM with 1000 mg/L D-Glucose

    Dulbecco's Modified Eagle's Medium (DMEM) with 1000 mg/L D-glucose

  3. Puromycin (Dihydrochloride)
    Puromycin

    Antibiotic; Protein synthesis inhibitor

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实验数据
产品说明书及文档
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概述

ReproRNA™-OKSGM是一种单链RNA复制子载体，包含五个重编程因子：OCT4、KLF-4、SOX2、GLIS1和c-MYC，以及一个嘌呤霉素耐药基因（图1）。这种RNA载体可以高效地将体细胞（如成纤维细胞）重编程为诱导多能干细胞（iPS），并且只需要一个转染步骤（图2）。当与ReproTeSR™重编程培养基一起使用时，iPS细胞集落的产生可以在无进料条件下实现，集落形态优越，重编程效率与基于进料的系统相似（图3 - 4）。ReproRNA™衍生的iPS细胞集落也表达未分化细胞的标记物，并保持正常的核型（图5-6）。随后，用ReproRNA™-OKSGM生成的iPS细胞可以在TeSR™维持培养基（mTeSR™1、TeSR™2或TeSR™-E8™）中维持，并进一步分化为所有三种胚层的细胞（图7）。

Subtype
Supplements
 
Cell Type
Pluripotent Stem Cells
 
Species
Human
 
Application
Cell Culture, Reprogramming
 
Brand
TeSR
 
Area of Interest
Stem Cell Biology
 

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Data Figures

Figure 1. Schematic of ReproRNA™-OKSGM, a Single‑Stranded RNA Replicon Vector

Figure 2. Schematic of Reprogramming Timeline with ReproRNA™-OKSGM

Somatic cells are transfected with ReproRNA™-OKSGM at day 0, and cultured in Advanced DMEM (AdvDMEM) with puromycin. After 6 days of puromycin selection post-transfection, cells are cultured in ReproTeSR™ for the remainder of the reprogramming induction phase until iPS cell colonies emerge. B18R recombinant protein is also added during the first 2 weeks after transfection to inhibit the interferon response and increase cell viability. Typically, by day 20, iPS cell colonies are large enough to be isolated and propagated in TeSR™ media. *TeSR™ = TeSR™ family media (mTeSR™1, TeSR™2, TeSR™-E8™).

Figure 3. ReproRNA™-OKSGM Vector Efficiently Reprograms Fibroblasts

Dermal fibroblasts were transfected with the ReproRNA™-OKSGM vector and reprogrammed under feeder-dependent (standard KOSR-containing hES cell medium on irradiated mouse embryonic fibroblasts (iMEFs)) or feeder-independent conditions (ReproTeSR™ on Corning® Matrigel®). Fibroblasts (passage 4) were reprogrammed with average efficiencies of 0.10 ± 0.03% (hES cell medium) and 0.20 ± 0.01% (ReproTeSR™). Reprogramming efficiency of fibroblasts with ReproRNA™ and ReproTeSR™ is comparable to that reported with Sendai virus.¹ (n ≥ 6; Data shown are mean ± SEM).

Figure 4. Feeder-Free Reprogramming with ReproRNA™-OKSGM Vector and ReproTeSR™ Generates iPS Cell Colonies with Superior Colony Morphology

Representative images of iPS cell colonies were generated using ReproRNA™‑OKSGM and cultured in (A) standard hES cell medium on irradiated mouse embryonic fibroblasts (iMEFs) or (B) ReproTeSR™ on Corning® Matrigel®. iPS cell colonies derived using ReproTeSR™ exhibit more defined borders, compact morphology, and reduced differentiation as compared to the ES cell medium.

Figure 5. Human iPS Cells Generated with ReproRNA™-OKSGM Express Undifferentiated Cell Markers

Human iPS cells generated with ReproRNA™-OKSGM display high expression of undifferentiated cell markers (OCT4 and TRA-1-60) as shown by flow cytometry analysis after 12 passages in mTeSR™1. (Filled histogram = sample, hollow histogram = secondary antibody only).

Figure 6. iPS Cells Derived Using ReproRNA™-OKSGM Display a Normal Karyotype

Karyogram of iPS cells derived with ReproRNA™-OKSGM and cultured in mTeSR™1 for 8 passages shows that a normal karyotype is retained.

Figure 7. ReproRNA™-OKSGM Derived iPS Cells Have the Capacity to Differentiate to Cells of the Three Germ Layers

Human iPS cells derived with ReproRNA™-OKSGM and maintained in mTeSR™1 for 7 passages were differentiated into cells of the three germ layers. Endoderm specification was achieved using the STEMdiff™ Definitive Endoderm Kit and flow cytometry analysis shows a high percentage of cells (98.7%) positive for endoderm markers (CXCR4+SOX17+). Mesoderm induction was achieved with STEMdiff™ Mesoderm Induction Medium as shown by the high percentage of cells (98.6%) expressing Brachyury (T). Ectoderm specification was demonstrated using STEMdiff™ Neural Induction Medium. CNS-enriched NPC cultures expressing PAX6 (green) and stained with DAPI (blue) are shown.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Information Sheet
Product Name
ReproRNA™-OKSGM Kit
Catalog #
05930
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
ReproRNA™-OKSGM
Catalog #
05931
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
ReproRNA™-OKSGM Kit
Catalog #
05930
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
ReproRNA™-OKSGM Kit
Catalog #
05930
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
ReproRNA™-OKSGM Kit
Catalog #
05930
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
ReproRNA™-OKSGM Kit
Catalog #
05930
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
ReproRNA™-OKSGM
Catalog #
05931
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
ReproRNA™-OKSGM
Catalog #
05931
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
ReproRNA™-OKSGM
Catalog #
05931
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
ReproRNA™-OKSGM
Catalog #
05931
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area
Workflow Stages

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Species Human
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