Heterodimeric expression of Oct4, Klf4, Sox2, and c-Myc transcription factors enables reprogramming of mouse somatic cells to induce functional stem cells. (1) Facilitates the re-differentiation of functional stem cells and makes full use of resources. (2) Reduces the risk of exogenous gene integration.

Operation method

protein transfection

Principle

1. It has been shown that a variety of proteins can enter cells by means of short peptide binding that can mediate protein transduction. 2. Various solvent-friendly and refolding techniques deal with the expression of inclusion body proteins There are biologically active proteins in Escherichia coli that have evolved to allow for the easy and large-scale production of therapeutic proteins.

Materials and Instruments

Mouse Fibroblasts
Valproic acid Culture medium
Petri dish Pipette gun Gun head Gun head box Refrigerator

Move

1. Each cycle of fibroblasts (with an initial cell density of 5 × ¹⁰⁴ cells/well, the first treatment was performed at night with recombinant reprogramming proteins (i.e., Oct4-11R, Sox2-11R, Klf4-11R, and c-Myc-11R), 1 mm valproic acid (VPA) supplied in either supplemented or supplemented mESC growth medium at 8 mg/ml, and an inhibitor of HDAC that can significantly increase the programming efficiency. ) protein, and an HDAC inhibitor that could significantly improve programming efficiency. The other medium was free of VPA and inhibitors, and both mediums were incubated for 36 h before the next cycle.

2. After completion of four repetitions of protein transduction, the bumped protein post-treated cells were transferred to irradiated mef-fed cells and briefly preserved in the mESC growth medium until cloned to 30-35 days.

3. The resulting mouse fibroblasts showed a stable and uniform expansion, and after more than 30 generations, rounded compact colonies were formed. They expressed pluripotent markers by immunochemical cell and staining.

Caveat

The required proteins and cells need to be kept at low temperatures to prevent inactivation.

Common Problems

1. iPSCs (especially for patients) are similar to ESCs but are easier to create in comparison, in this case are human cells, and are less controversial, presenting biomedical unprecedented opportunities for medical research and clinical applications. Achieved iPSCs require improved guidance methods for differentiation to generate populations of homogeneous lineage-specific cell types.

2. utilizing cellular endogenous gene expression strategies also allows for easier reprogramming of desired exogenous genes.

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