To address this, we attempted to generate rat iPS cells by introducing total ESC-extracts into permeabilized rat NP cells.
Based on previous studies showing that fusion of ESCs with somatic cells induce nuclear reprogramming [23], [24], [25], we hypothesized that introduction of ESC total proteins into rat somatic cells may reprogram them to a pluripotency.
However, the proportion of polyA RNA purified from ESMV and ESC total RNA (0.59% and 3.43%) reflected a 5.8-fold difference, whereas a much larger reduction of at least 55-fold was observed with all transcripts tested by quantitative RT-PCR.
cDNA was synthesized with 1 µg of total RNA from ESCs using the ImPromII Reverse Transcription System (Promega) and random primers.
In this study, we sought to establish an efficient procedure to generate iPS cells from two different rat tissues, neural precursors (NPs) and rat embryonic fibroblast (REF), by introducing total extracts from ESCs and/or retroviral transduction of defined transcription factors.
Total RNA from ESCs was isolated with Tri reagent (Sigma) and further purified with the Qiagen column kit (Qiagen, Milan, Italy).
A total of 10 ESCs (R1, FLAG Ctrl, FLAG MKRN1, shGFP, or shMKRN1) were seeded on sterilized, 0.1% gelatin-coated glass coverslips (Corning; No. 1 22 mm sq) and cultured for 48 h in mESC media.
In this study, a total of seven putative ESC lines were derived from porcine embryos of various origins, including in vitro fertilization, parthenogenetic activation, and, in particular, induced pluripotent stem (iPS) nuclear transfer (NT) from a donor cell with induced pluripotent stem cells (iPSCs).
Consistent with their functional role in ESC, a total of 84,163 primate-specific TF-binding sequences for NANOG, POU5F1.
Unlike total RNA collected from ESCs, the total RNA from ESMVs does not contain 28S and 18S rRNA (Figures 1A and 1B).
