ReproRNA™-OKSGM

A non-integrating, self-replicating RNA reprogramming vector for generating iPS cells
Catalog #
05930_C
A non-integrating, self-replicating RNA reprogramming vector for generating iPS cells
From: 1,463 USD
Required Products
  1. Puromycin (Dihydrochloride)
    Puromycin

    Antibiotic; Protein synthesis inhibitor

  2. Trypsin-EDTA (0.25%)
    Trypsin-EDTA (0.25%)

    Enzymatic cell dissociation reagent

  3. 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

Overview

ReproRNA™-OKSGM is a single-stranded RNA replicon vector that contains five reprogramming factors: OCT4, KLF-4, SOX2, GLIS1, and c-MYC, as well as a puromycin-resistance gene (Figure 1). This RNA vector reprograms somatic cells, such as fibroblasts, into induced pluripotent stem (iPS) cells with high efficiency and only requires a single transfection step (Figure 2). When used together with ReproTeSR™ reprogramming medium, the generation of iPS cell colonies can be achieved under feeder-free conditions with superior colony morphology and similar reprogramming efficiency to feeder-based systems (Figures 3 - 4). ReproRNA™-derived iPS cell colonies also express markers of undifferentiated cells and retain a normal karyotype (Figures 5-6). Subsequently, iPS cells generated with ReproRNA™-OKSGM can be maintained in TeSR™ maintenance media (mTeSR™1, TeSR™2, or TeSR™-E8™) and further differentiated into cells of all three germ layers (Figure 7).
Advantages
• Non-viral, non-integrating vector system
• Self-replicating vector only requires a single transfection
• Vector contains all reprogramming factors
• Comparable fibroblast reprogramming efficiency to Sendai virus
Components
  • ReproRNA™-OKSGM Kit (Catalog #05930)
    • ReproRNA™-OKSGM vector (Catalog #05931)
    • ReproRNA™ Transfection Reagent
    • ReproRNA™ Transfection Supplement
    • Recombinant B18R Protein (Catalog #78075)
 
Subtype
Supplements
Cell Type
Pluripotent Stem Cells
Species
Human
Application
Cell Culture, Reprogramming
Brand
TeSR
Area of Interest
Stem Cell Biology

Scientific Resources

Product Documentation

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
Product Name
ReproRNA™-OKSGM
Catalog #
05931
Lot #
All
Language
English

Educational Materials (5)

Brochure
2019-2020 Cell Culture Training Catalog
Brochure
Products for Human Pluripotent Stem Cells
Brochure
ReproRNA™-OKSGM Non-Integrating Reprogramming Vector
Wallchart
Derivation and Applications of Human Pluripotent Stem Cells
Video
How to Reprogram Fibroblasts into Human Induced Pluripotent Stem (iPS) Cells Using ReproRNA™-OKSGM
5:06
How to Reprogram Fibroblasts into Human Induced Pluripotent Stem (iPS) Cells Using ReproRNA™-OKSGM

Product 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.

Data and Publications

Data

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.

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