Generate Mouse IPS Cells Line By Three Factors Under Feeder-Free Condition

Takahashi and Yamanaka reported their milestone strategy to reprogramme MEF cells to ES cells-like cells by overexpression of four transcription factors: Oct4, Sox2, c-Myc and K1f4. iPS cells are highly similar to ES cells counterparts_morphologically, the level of gene expression, pluripotency, the ability of differentiation, genome-wide distribution of chromatin modifications, and have no argue of ethics. The technical of iPS cells generation have acquire a lot of achievement because of its enormous process in clinical. However, a majority of iPS cells generation still use MEF cells as feeder layers. Because there is exogenous material, zoonotic pathogens, unknown viruses in mouse feeder cells, so it will hinder clinical translation of iPS cells. And when screening the intermediates iPS cells the feeder cells would be mixed with the iPS cells samples, it would affect the study of the cellular reprogramming mechanisms. Here we introduce a method to generate mouse iPS cells use three transcription factors Oct4, Klf4and Sox2under feeder-free conditions, we add Vitamin C after Virus infection and gradually replaced the medium, made the reprogramming efficiency increase to0.2%.Objective Generate mouse iPS cells lines by three factors under feeder-free condition, at the same time master the iPS cells technology, and build a base for investigate the reprogramming mechanisms and used for clinical medicine.Methods (1) The vectors of pMXs-Oct4, pMXs-Sox2and pMXs-Klf4were transfected into plat-E cells, generate Virus that encoding the mouse reprogramming genes of Klf4, Oct4andSox2.(2) MEF cells were incubated with Viral-containing supernatants for Sox2, Oct4and Klf4under feeder-free condition, through add Vitamin C after Virus infection and gradually replaced the medium to increase reprogramming efficiency.(3)Through morphology, pluripotency gene expression analysis, Teratomas assays, hematoxylin-eosin staining to detect the iPS cells line.Results (1) we succeed generate iPS cells from MEF cells by three factors under feeder-free condition, and the iPS cells are molecularly and functionally similar to ES cells.(2) Through add Vitamin C and gradually replaced the medium after retroviral infection, we promote the efficiency of induced iPS cells to0.2%.