Novel codon-optimized mini-intronic plasmid for efficient, inexpensive and xeno-free induction of pluripotency

• Published in: Scientific reports 2015-01, Vol.5 (1), p.8081-8081, Article 8081
• Main Authors: Diecke, Sebastian, Lu, Jiamiao, Lee, Jaecheol, Termglinchan, Vittavat, Kooreman, Nigel G., Burridge, Paul W., Ebert, Antje D., Churko, Jared M., Sharma, Arun, Kay, Mark A., Wu, Joseph C.
• Format: Article
• Language: English
• Subjects: 13/100; 13/109; 13/44; 13/51; 42; 42/35; 631/1647/2300/1514; 631/532/2435; Animals; c-Myc protein; Cells, Cultured; Cellular Reprogramming; Codon; Color; Drug screening; Epstein-Barr virus; Epstein-Barr Virus Nuclear Antigens - genetics; Epstein-Barr Virus Nuclear Antigens - metabolism; Fibroblasts; Fibroblasts - cytology; Fibroblasts - metabolism; Genomes; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Humanities and Social Sciences; Humans; Immunity, Innate; Integration; Introns; Karyotyping; KLF4 protein; Leukocytes (mononuclear); Leukocytes, Mononuclear - cytology; Leukocytes, Mononuclear - metabolism; Mice; Microscopy, Fluorescence; multidisciplinary; Myc protein; Nanog Homeobox Protein; Oct-4 protein; p53 Protein; Peripheral blood mononuclear cells; Plasmids - genetics; Plasmids - metabolism; Pluripotency; Pluripotent Stem Cells - cytology; Pluripotent Stem Cells - metabolism; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Regenerative medicine; Ribonucleic acid; RNA; RNA, Small Interfering - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Rodents; Science; Stem cells; Transcription Factors - genetics; Transcription Factors - metabolism; Transfection; Tumor Suppressor Protein p53 - antagonists & inhibitors; Tumor Suppressor Protein p53 - genetics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep08081

The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2 and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.