File:Rat induced pluripotential stem cell protocol.jpg: Difference between revisions

Generation of rat Induced Pluripotent Stem Cells (iPS) cells from rat embryonic fibroblasts

To generate germline-competent riPSCs, we initially infected Wistar (WI) or Dark Agouti (DA) rat embryonic fibroblasts (REFs) from embryonic day 14.5 or 15.5 (E14.5 or E15.5) with a lentiviral vector carrying three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) controlled by a tetracycline-responsive regulatory element and with Ubc-promoter - driven reverse tet transactivator (rtTA) and EGFP (Fig. 1A). We added doxycycline (Dox) to the culture medium on the day of infection (day 0), with rat LIF (rLIF) added to medium on day 1

(A) Schema of inducible lentivirus vector. XbaI enzyme restriction site exists between Ubiquitin-C (Ubc) promoter and reverse tet transactivator (rtTA) element.

(B) Schematic time schedule of riPSCs generation.

(C)–(D) Morphology of generated riPSCs (passage P0); bright field and fluorescent field (C). ALP staining after clonal passage (D).

Figure 1. Journal.pone.0022008.g001.jpg doi:10.1371/journal.pone.0022008.g001

Reference

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0022008

© 2011 Hamanaka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.