ReproRNA™-OKSGM

一种非整合型、自我复制的 RNA 重编程载体，用于生成诱导多能干细胞

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产品号 #05930_C

一种非整合型、自我复制的 RNA 重编程载体，用于生成诱导多能干细胞

产品优势

非病毒、非整合型载体系统
自我复制载体，仅需一次转染
载体包含所有重编程因子
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（目录号 #05930）
ReproRNA™-OKSGM 载体（目录号 #05931）

ReproRNA™ 转染试剂
ReproRNA™ 转染补充剂
重组 B18R 蛋白（目录号 #78075）

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总览

ReproRNA™-OKSGM 是一种单链 RNA 复制子载体，包含五种重编程因子：OCT4、KLF-4、SOX2、GLIS1 和 c-MYC，以及一个嘌呤霉素抗性基因（见图 1）。该 RNA 载体能够高效地将体细胞（如成纤维细胞）重编程为诱导多能干细胞（iPS 细胞），且仅需一次转染步骤（见图 2）。

当与 ReproTeSR™ 重编程培养基联合使用时，可在无饲养层条件下成功生成 iPS 细胞克隆，具有优异的克隆形态，且重编程效率与传统有饲养层系统相当（见图 3 - 4）。由 ReproRNA™ 重编程产生的 iPS 细胞克隆会表达未分化细胞标志物，并保持正常的核型（见图 5 - 6）。

随后，用 ReproRNA™-OKSGM 生成的 iPS 细胞可在 TeSR™ 系列维持培养基中（如 mTeSR™1、TeSR™2 或 TeSR™-E8™）继续培养，并可进一步分化为三胚层来源的细胞类型（见图 7）。

实验数据

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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ReproRNA™-OKSGM Kit

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