A successful reprogramming strategy using
episomal vectors was recently described, making keratinocytes even more interesting in the future of iPSC generation [74].
Primer Sequence Vector EBNA_1 forward 5'-TTTAATACGATTGAGGGCGTCT-3' pEB-C5 EBNA_1 reverse 5-GGTTTTGAAGGATGCGATTAAG-3' pEB-C5 Tg foward 5-GCCAGGTGGGTTAAAGGAGC-3' pEB-Tg Tg reverse 5-GGTACTTATAGTGGCTGGGCTGT-3' pEB-Tg Primer [micro]g DNA Ta ([degrees]C) EBNA_1 forward 0,1 57 EBNA_1 reverse Tg foward 0,02 60 Tg reverse TABLE 2: Efficiency of reprogramming of MNCs by
episomal vectors, showing the details of the 6 experiments.
E14T cells were electroporated with an
episomal plasmid containing the genes for puromycin resistance under Pvec and hygromycin resistance under Thymidine Kinase promoter (Figure 1(a)).
Skin fibroblasts were reprogrammed into iPSCs by Sendai viral transduction of four
episomal vectors carrying KLF4, OCT3/4, SOX2, and c-MYC (Figures 1(a) and 1(b)).
Thus, iPSCs generated from donor cells using nonintegrative technologies (mRNA, Sendai,
Episomal, etc.) could be a promising source of osteogenic cells [20-23].
Chen et al., "Generation of integration-free induced pluripotent stem cells from human peripheral blood mononuclear cells using
episomal vectors," Journal of Visualized Experiments, no.
In order to overcome problems associated with mutagenesis resulting from ectopic gene insertion, adenoviral,
episomal, or plasmid vectors were used as alternatives in the course of developing the next generation of reprogramming methods [84, 85].
Citation: "Efficient Feeder-Free
Episomal Reprogramming with Small Molecules;" Junying Yu, Kevin Fongching Chau, Maxim A Vodyanik, Jinlan Jiang, Yong Jiang; PLoS ONE, published 1 Mar 2011, 10.1371/jounial.pone.0017557.
Sendai virus,
episomal vectors, small molecules, proteins, and modified RNAs are commonly employed to avoid integration [169-171].
The
episomal marker is located within the ORF 470 of the 35-kb plastid (11), which is physically unlinked to genomic markers.
The hiPSC line from donor 2 (male, here abbreviated as D2) was generated using the integration-free
episomal 4-in-1 CoMiP reprogramming plasmid (Addgene, #63726) with the reprogramming factors OCT4, KLF4, SOX2, and c-MYC and short hairpin RNA against p53, as described previously with modifications [25].
Life Technologies' commercialization of Essential 8 Medium, Vitronectin (VTN-N), and
Episomal iPSC Reprogramming Vectors addresses several challenges associated with developing relevant cells for use in a wide range of studies, from basic and translational research to drug discovery efforts.