Generation of induced pluripotent stem cells using site-specific integration with phage integrase

Abstract

To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ΦC31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene expression, and epigenetic states similar to ES cells and formed teratomas with three germ layers in nonobese diabetic/severely compromised immunodeficient mice. Importantly, these iPS cells have only a single integration site in each cell line. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. The effect of the insertion on the expression of these genes can be studied by RT-PCR. No insertion into microRNA gene loci was detected. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. We conclude that phage integrase-mediated sitespecific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application.

Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes

“…This is supported by the fact that different transcription factor combinations with the addition of small molecules have differing effects on iPSC generation (14)(15)(16)(17)(18)(19). Recent studies have demonstrated nontranscription factor-mediated reprogramming of somatic cell into iPSCs based solely on vector transfection, revealing the complexity and diversity of the underlying mechanisms of cell reprogramming (20,21). Selected individual genes from single cells demonstrated a wide range of expression levels in all cell types (Fig.…”

Generation of iPS Cells Using a BacMam Multigene Expression System

“…Using autonomous self-cleaving 2A peptides is also a method to avoid the transcriptional interference and achieve stoichiometric expression of multiple genes from a single promoter (Szymczak et al, 2004). Although polycistronic vectors with 2A-linked reprogramming factors have been widely used in generating iPS cells both in viral and non-viral methods (Carey et al, 2009;Kaji et al, 2009;Sommer et al, 2009;Woltjen et al, 2009;Ye et al, 2010), non-integrating methods for generating iPSC using plasmid, oriP/EBNA-1 (Epstein-Barr nuclear antigen-1)-based episomal and 'minicircle' vectors (Okita et al, 2008;Yu et al, 2009;Jia et al, 2010), still have generally suffered from low reprogramming efficiencies, mostly due to low transduction rates against the primary somatic cells. Reprogramming by adenoviral vectors has been expected to overcome the problem of low transduction efficiencies of nonintegrating vectors, the efficiency of reprogramming was, however, still low (Stadtfeld et al, 2008;Zhou and Freed, 2009).…”

Generation and Genetic Engineering of Human Induced Pluripotent Stem Cells Using Designed Zinc Finger Nucleases

“…Generation of iPSCs from human and murine cells using other similar methods such as piggybac transposons, phage integrase (FC31), and excisable lentiviruses have been reported elsewhere [32,33]. Although the FC31-derived hiPSCs had only a single integration in each line, the locations of integration were random in different lines, favoring intergenic regions [32]. It is quite possible that site-specific integration using FC31 integrase could result in insertions at critical genes or control regions in targeted cells disrupting normal function of these cells.…”