Generation of Transgenic Porcine Chimeras using Primordial Germ Cell-Derived Colonies1

Abstract

In mice, two pluripotent cell lines, embryonic stem (ES) cells and embryonic germ (EG) cells, have been identified. We present here results indicating that porcine EG cell lines can be isolated, genetically transformed, and utilized to make transgenic chimeras. Briefly, primordial 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 medium supplemented with 0.01 mM nonessential amino acids, 2 mM glutamine, 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 40 ng/ml human stem cell factor, 20 ng/ml human basic fibroblast growth factor, and 20 ng/ml human leukemia inhibitory factor. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein under control of the cytomegalovirus promoter. After electroporation, cells were plated and later examined under fluorescein isothiocyanate excitation. Fluorescent colonies were selected for chimera generation. Blastocysts collected from gilts on Day 5 were injected with 10-15 transgenic PGC-derived cells and transferred into recipient gilts. Gilts were hysterectomized on Day 25, and fetal tissues were analyzed by Southern blotting. Three chimeras out of 20 fetuses analyzed were transgenic. Additionally, when one recipient gilt was allowed to go to term, one piglet with transgenic contribution was identified.

The culture and establishment of embryonic germ (EG) cell lines from Chinese mini swine

“…In the present study, the three growth factors were generally used for culturing pig PGC/ EG cells. However, we did find that in the initial culture, PGCs could also proliferate on STO feeder layer alone without the supplement of growth factors, as reported by Shim et al and Piedrahita et al [11,12]. In the case of long-term culture experiment (successive passaging), it is better to add SCF in culture medium, unless fresh feeder layer is frequently replaced.…”

“…Karyotype analysis was done by the method of Mueller et al [12] with modification. At passage 18-20, EG cells plated on gelatin-coated 25 cm2 flasks were cultured for 48 h, and treated with 0.5 μg/ml colcemid (Sigma) for 5 h at 37 o C in CO 2 .…”

“…Under appropriate in vitro conditions, these PGCs derived mouse embryonic germ cells (EG cells) give a close morphological and developmental resemblance to ES cells [8][9][10]. Several attempts to establish pluripotent PGCs-derived cell lines in pig have also been described and the injection of either PGC cells or shortterm cultured EG cells into recipient blastocysts and then transferred into surrogate mothers did get living chimeric offspring but without germ line transmission [11][12]. However, Mueller et al has demonstrated the presence of injected EG cells in gonad tissue of one chimeric fetus by PCR and blot methods [13].…”

Transgenic bovine as bioreactors: Challenges and perspectives

“…38,39 However, the establishment of pluripotent ESCs from bovine embryos has proven unsuccessful, and the generation of transgenic bovine chimeric offspring was only possible using nuclear transplantation, where transgenic embryonic stem (ES)-like cells were produced from modified fetal bovine fibroblasts. 40 Transgenic porcine chimeras were generated using primordial germ cell (PGCs)-derived colonies, 41 and PGCs can become EGCs by in vitro or in vivo differentiation. Induced pluripotent stem cells (iPSCs) are stem cells that can be generated via cellular reprograming using gene transduction or recombinant protein treatment of somatic cells.…”

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

“…Alkaline phosphatase (AP) activity was determined essentially as described by previous study (Piedrahita et al 1998;Niu et al 2013). …”