Results: 3

1.

Figure 2. Pluripotency of iPSC before and after Cre-mediated excision. From: Site-specific recombinase strategy to create iPS cells efficiently with plasmid DNA.

(A) Quantitative RT-PCR data showing expression of Klf4, cMyc, GFP, Oct4, Sox2, Rex1, and Nanog in MEF- and ASC-iPSC before and after removal of the reprogramming cassette, as well as in the parental MEF and ASC and in ESC controls. (B) Promoter methylation status of Oct4 (left panel) and Nanog (right panel) in iPSC and iPSC-X lines. Five different CpG islands for each line were analyzed, indicated by their distance to the respective transcription start site (TSS). Open circles reflect low methylation (0–25%), whereas gray circles represent medium (26–75%) and black circles high (76–100%) methylation. (C) Immunofluorescence staining of Oct4, SSEA1, and EGFP in MEF- and ASC-iPSC before and after Cre recombinase treatment. Scale bars represent 50µm.

Marisa Karow, et al. Stem Cells. 2011 November;29(11):1696-1704.
2.

Figure 3. In vivo studies of pluripotency of iPSC before and after Cre-mediated excision. From: Site-specific recombinase strategy to create iPS cells efficiently with plasmid DNA.

(A) Day 14 embryoid bodies were stained with the antibodies anti-SMA, anti-Tuj1, or anti-AFP to show mesodermal, ectodermal, and endodermal differentiation in vitro, respectively. Alexa 594-labeled secondary antibodies were used, while Hoechst staining was used to visualize the nuclei. Figure represents merged pictures. Scale bars represent 50 µm. (B) Histological samples obtained 4 weeks after injection of MEF-iPSC (left panel) or ASC-iPSC (right panel) into SCID beige mice showing teratomas composed of cells with mesoderm, ectoderm, and endoderm lineages. (C) Chimeric pups obtained after injection of MEF-iPSC (left image) and MEF-iPSC-X (right image) into blastocysts of albino B6 mice. iPSC gave rise to black fur.

Marisa Karow, et al. Stem Cells. 2011 November;29(11):1696-1704.
3.

Figure 1. Generation of iPSC and removal of reprogramming factors using site-specific recombinases. From: Site-specific recombinase strategy to create iPS cells efficiently with plasmid DNA.

(A) Schematic overview of the recombinase strategy3/29/2011. First, ϕC31 integrase is used to integrate the p4FLR plasmid at a preferred location and reprogram somatic cells. A clone with a single copy of the plasmid integrated at a safe, intergenic location is chosen. Second, Cre recombinase mediates precise excision of the reprogramming cassette. (B) SSEA1 staining of an ASC-iPSC on day 20 after nucleofection before picking (upper panel) and morphology of established iPSC lines generated from MEF and ASC starting populations shown by alkaline phosphatase staining compared to ESC (lower panel). Scale bars represent 50µm. (C) Southern blot analysis of representative MEF- and ASC-iPSC lines before and after Cre-mediated excision of the reprogramming cassette, by using an EGFP probe. Both clones carried a single integration of the reprogramming cassette, which was no longer detectable after excision. (D) Verification of the genomic integration site determined previously by LM-PCR using pairs of the respective genomic and plasmid-binding primers for both of the iPSC clones as depicted schematically (right panel). Genomic DNA of the parental cells was used as a negative control, proving specific product amplification (left panel). Cre-mediated excision of the reprogramming cassette was verified by amplification of the genomic integration locus (left panel). Genomic DNA of cells bearing the entire cassette served as negative controls, since in those samples the small PCR product could not be detected by using a combination of primers binding adjacent to the genomic integration site and within the recombined sites (dashed lines in right panel). PCR to test for pVI plasmid (lower left panel) showing the absence of the plasmid in established iPSC lines. Ctrl = control.

Marisa Karow, et al. Stem Cells. 2011 November;29(11):1696-1704.

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