Classification Biological Sciences/ Agricultural Sciences Keywords primitive endoderm, stem cell, chimera, XEN, SCNT Author Contributions CP, JCB and BT conceived the project and designed the experiments; CP, YJ, YU and JB established and performed characterization of embryonic outgrowths and XEN cells; KP and AP performed the knockin and generated the GFP reporter lines used for generating GFP:XEN cells used in chiera experiments; CP, JL, LP, TL, JS, YG, YS, JW 2 generated the libraries and transcriptomic analysis; CP and BT wrote the initial draft, CP, JW, JCB and BT revised the manuscript based on the input from all authors. All authors approved final draft for submission. Abstract In this article, we report for the first time the derivation and characterization of extraembryonic endoderm (XEN) cells from primitive endoderm (PrE) of porcine (p) embryos. The pXEN cells can be reliably and reproducibly generated from parthenote, in vitro and in vivo derived embryos. The pXEN cells retained all the hallmarks of PrE including expression of canonical PrE and XEN cell markers (GATA4, GATA6, SOX17, SALL4, FOXA2, and HNF4A). Transcriptome analysis further confirmed their XEN cell origin. The pXEN cells when introduced into blastocyst stage embryo contributed to widespread chimerism including visceral yolk sac, chorion, as well as embryonic gut and liver primordium in the fetus. The pXEN cells were shown to be an efficient nuclear donor for generating cloned offspring. Taken together, pXEN cells fulfil a longstanding need for a stable, chimera-competent, and nuclear transfer-compatible porcine embryonic cells with applications for agriculture and medicine. Significance StatementWe report for the first time, the derivation and characterization of extraembryonic endoderm (XEN) stem cells from porcine (p) embryos. The pXEN cells can be reliably and reproducibly derived from primitive endoderm precursors. When injected into blastocyst-stage embryos, the pXEN cells have contributed to wide-spread chimerism including visceral yolk sac, chorion of the extraembryonic membranes, as well as definitive endoderm of the fetus, primarily the embryonic gut and liver primordium.Additionally, these XEN cells have proven to be an efficient nuclear donor for generating cloned offspring. These newly discovered stem cells provide a novel model for studying 4 lineage segregation, as well as a source for interspecies chimeras for generating endodermal organs, and for genome editing in livestock. 5