generation of induced pluripotent stem cells

( D) Homology scores between the 39-nt minimal wild-type attP site (20), or between 28-nt consensus pseudo attP sequences (23), and the 50-nt genomic sequences spanning the insertion site of the five mouse and three human chromosomes described in Table 1. Note that mIntOS16 and mIntOK2 have identical sequences. The positions of the primers P1 and P2 used to sequence the right and left arms, respectively, are indicated in Fig.1 A. ( C) Sequences of the regions adjacent to the insertions in mouse and human AF iPS cells. Although these two insertions into chromosome 14 have identical flanking sequences, the differences in the patterns of the two clones caused by the different positions of the KLF4 gene confirm that they were two separate insertion events. Mouse iPS cell clones mIntOS16 and mIntOK2 integrated with the OSKM and OKSM constructs, respectively, are compared with the plasmid (OS and OK) constructs. ( B) Digestion of the Sph1 site at the KLF4 gene. The absence of 4.3- and 2.7-kb bands in the clones indicates nonspecific integrations. One or more extra bands are generated as the result of the absence or presence of BamH1 site within the recircularized genomic DNA. Specific integrations are revealed by the 4.3- and 2.7-kb bands from the three BamH1 sites in the expression cassette and are represented in the control OKSM plasmid construct (OK). ( A) Fragment size of rescued plasmids from mouse ( Left) and human ( Right) iPS cells. Studies of the integration sites of the mouse and human AF iPS cells. Note that a single copy also can be seen in an interphase nucleus. ( Right, Inset) The red arrow indicates the chromosome location of the integration site by reverted DAPI G-banding in situ and ( Left, Inset) by the green arrow with chromosome ideogram. The white arrow indicates the insertion site in a mouse metaphase. ( E) FISH showing a single-copy insertion in chromosome 14, band 14B, using the rtTA probe (mIntOK2 is shown). Single bands larger than 10 kb indicate single insertions into the genome. The endogenous bands are marked by arrows. ( D) Southern blot analysis of DNA prepared from iPS cells generated from MEF after digestion with Ssp1, which cuts the construct once.

(The abbreviation “int” denotes clones obtained using integrase.) ( C) Teratoma formation in immunodeficient mice by these two clones. Two of the integrase-generated mouse iPS clones, mIntOK3 and mIntOS16, from MEF with OKSM and OSKM respectively, are compared with mouse ES cells. ( B) Pluripotency markers Nanog and SSEA1. The locations of the two probes to detect the inserts on Southern blots are shown. B, BamH1 P1 and P2, primers used for sequencing the integration sites. The constructs then are joined to the plasmid pJTI-Fast-DEST (Invitrogen) at the Sph1 and Sac1 sites. The Advanced Tet-On system consists of a CAG promoter driving the transactivator rtTA linked to the chicken β-globin insulator-flanked TRE-Tight promoter driving the polycistronic OSKMG or OKSMG (labeled “OS” and “OK,” respectively, in this and subsequent figures). ( A) The two composite constructs for integration. We conclude that phage integrase-mediated site-specific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. No insertion into microRNA gene loci was detected. The effect of the insertion on the expression of these genes can be studied by RT-PCR. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. Importantly, these iPS cells have only a single integration site in each cell line. 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. Here, we describe a method using bacteriophage ΦC31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. 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.

Generation of induced pluripotent stem cells