GENERATION OF TRANSGENIC PORCINE CHIMERAS USING PRIMORDIAL GERM CELL-DERIVED COLONIES

In mice, two pluripotent cell lines, embryonic stem (ES) cells and emb ryonic germ (EC) cells, have been identified. We present here results indicating that porcine EC cell lines can be isolated, genetically tra nsformed, and utilized to make transgenic chimeras. Briefly, primordia l germ cells (PGCs) were isolated from Day 25-27 fetuses and plated on STO feeder cells in Dulbecco's modified Eagle's medium:Ham's F-10 med ium supplemented with 0.01 mM nonessential amino acids, 2 mM glutamine , 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 40 ng/ml human ste m cell factor, 20 ng/ml human basic fibroblast growth factor, and 20 n g/ml human leukemia inhibitory factor. For genetic transformation, cel ls were electroporated with a construct containing the green fluoresce nt protein under control of the cytomegalovirus promoter. After electr oporation, cells were plated and later examined under fluorescein isot hiocyanate excitation. Fluorescent colonies were selected for chimera generation. Blastocysts collected from gilts on Day 5 were injected wi th 10-15 transgenic PGC-derived cells and transferred into recipient g ifts. Gilts were hysterectomized on Day 25, and fetal tissues were ana lyzed by Southern blotting. Three chimeras out of 20 fetuses analyzed were transgenic. Additionally, when one recipient gut was allowed to g o to term, one piglet with transgenic contribution was identified.