Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2015 Mar 12;4(4):727–743. doi: 10.1016/j.stemcr.2015.02.004

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2015 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

PMC Copyright notice

KLF4 Isoforms Underlie Phenotypic Differences in Reprogramming

(A) Diagram of the PB reprogramming system and analysis steps. The PB-TAC transposon delivers inducible, reporter-linked (mCherry), polycistronic reprogramming cassettes (Poly) into ROSA-rtTA; Nanog-GFP MEFs (d-1). Cultures are induced with dox (d0, dox; filled arrows) and harvested on d8 for FACS and passage for late-stage analysis (d18). For AP staining, cultures are maintained without passage until d10. Full reprogramming and transgene independence is assessed by dox withdrawal from d18 to d24 (open arrows). Predicted expression periods for the mCherry and GFP reporters during iPSC derivation are shown with red and green gradated bars. Blue polygons represent PB 3′ (left) and 5′ (right) inverted terminal repeats. tetO, dox-responsive promoter; IRES, internal ribosome entry signal; pA, polyadenylation signal.

(B) AP staining on d10 of OSKM and OKMS reprogramming cultures. Scale bar, 4,000 μm.

(C) Day 18 fluorescence microscopy of entire wells (composite 10 × 10 fields) or selected insets (3 × 3 subfields) for mCherry⁺ and Nanog-GFP⁺ (left). Scale bars, 4,000 μm (full well) and 1,000 μm (inset). FACS analysis of mCherry and Nanog-GFP expression in d18 Total and SSEA-1⁺ populations (right).

(D) Polycistronic cassette structure and sequence of the 2A-Klf4 N-terminal cleavage junctions. Cloned cDNA is compared with murine Klf4 GenBank mRNA sequences (U20344.1 and U70662.1), indicating the position of the predicted initiation codons and amino acid translations. The N-terminal 9aa of U20344.1 were introduced into OKMS (Klf4_S) to produce OK⁺⁹MS (Klf4_L).

(E and F) The OK⁺⁹MS construct was evaluated according to the assays outlined in (A)–(C). The results in (B), (C), (E), and (F) are representative of the results from at least three independent experiments (summarized in Figures S1A and S1B).

(G and H) Time-course analysis of mCherry⁺ cell expansion (G) and SSEA-1⁺ fractions (H) from OSKM, OKMS, and OK⁺⁹MS transfections cultured for the indicated number of days before (2, 4, 6, 8) and after (10, 14, and 18) passage. Means ± SE for three independent experiments.

(I and J) Time-course analysis of mCherry silencing (left) and Nanog-GFP activation (right) in SSEA-1⁺ cells on the indicated number of days after day 8 passage (10, 14, and 18). Means ± SE for three independent experiments.

See also Figure S1.