Efficient generation of mouse ESCs-like pig induced pluripotent stem cells

Dear Editor, Porcine induced pluripotency stem cells (piPSCs) are promised in basic research, animal husbandry and regenerative medicine. However, the efficiency of the piPSCs induction has been low and the generated piPSCs varied in cell morphology and cell characteristics. Here we report a novel approach to improve efficiency of piPSCs generation. The induced piPSCs are dome-shaped mouse embryonic stem cells (ESCs)-like and display molecular properties of mouse ESCs. Electroporation study reveals that mouse ESCslike status facilitates genetic manipulating of piPSCs. Importantly, we demonstrate that the domed piPSC colonies are more suitable as donor cells for nuclear transfer (NT) to generate reconstructed embryos than those flattened piPSCs. The potential applications of the newly generated piPSCs in ungulate pluripotent research are discussed.

Finally, 10 4 PEFs per well were infected with concentrated virus in a 24-well plate. When the PEFs reached 90% confluence, they were split into 12-well plates pre-seeded with feeders.
On the second day, the media were replaced by KOSR or LBX.

Transgenic manipulation of piPSCs
When carrying out transgenic manipulation, the piPSCs were digested into single cells. 110 6 cells were electroporated with 6 μg plasmid carrying a PGK-neo r cassette and 2 μg plasmid carrying a transposase cassette by Neon Transfection System (Invitrogen) at 2400 V, 20 ms and two pulses according to the manufacturer's instructions. Then the cells were plated onto 60-mm dishes coated with G418-resistant feeders and filled with the culture medium containing 250 μg/ml G418 for selection of resistant cells. After 4-6 days, G418-resistant colonies were selected for sub-cell-line derivation.

Karyotype analysis
Karyotype analysis was performed as previously described (Zhao et al., 2009).

Embryoid body formation
PiPSCs were digested into single cells and transferred into gelatin-coated plates followed by incubating in DMEM medium for 10 min to discard the feeder cells. Then the suspensions were harvested and transferred to a dish containing KOSR medium lacking bFGF. The medium was changed every other day and on day 6 the EBs were collected.

Teratoma formation and histological analysis
After digesting, the piPSCs were transferred to gelatincoated plates to discard the feeder cells.
Then, collect the cells for centrifuge. Next, suspend the cell pellets using PBS and 300 μl suspension containing 1×10 7 piPSCs were injected under the skin of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. The teratomas could be harvested for histological analysis at 4 weeks. And then the tumors are fixed and sliced.
Sections were stained with haematoxylin and eosin.

RT-PCR and quantitative PCR analyses
Total RNA was extracted using Trizol reagent (Invitrogen). RT-PCR was performed by using Q-PCR SYBR Green Real-time PCR Master Mix (Toyobo), High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, 4368814) and Real-time PCR equipment (Agilent Mx3005P) according to manufacturer's instructions. Electrophoresis was performed on a 2% agarose gel. All primers were listed in Table S3.

Statistical Analysis
PiPSCs colony numbers and quantitative PCR results were analyzed by analysis of variance (ANOVA) with P< 0.05 considered significant.