Activating the Expression of Human K-rasG12D Stimulates Oncogenic Transformation in Transgenic Goat Fetal Fibroblast Cells

• Published in: PloS one Vol. 9; no. 3; p. e90059
• Main Authors: Gong, Jianhua, Wang, Zhongde, Polejaeva, Irina, Salgia, Ravi, Kao, Chien-Min, Chen, Chin-Tu, Chen, Guangchun, Chen, Liaohai
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 2014; Public Library of Science (PLoS)
• Subjects: Activation; Adrenal cortex; Agriculture; Animal models; Animals; Animals, Genetically Modified; Biochemistry; Biology; Cancer; Carcinogenesis - metabolism; Carcinogenesis - pathology; Cell proliferation; Cell Transformation, Neoplastic - metabolism; Cell Transformation, Neoplastic - pathology; Cloning; Disease; Engineering; Enzymes; Fetus - pathology; Fetuses; Fibroblasts; Fibroblasts - metabolism; Fibroblasts - pathology; Gene expression; Genetic engineering; Genetic transformation; Genetic Vectors - metabolism; Genomes; Goats; Goats - genetics; Green Fluorescent Proteins - metabolism; Herpes simplex; Herpes viruses; Herpesvirus 1, Human - metabolism; Homologous recombination; Homology; Humans; Integrases - metabolism; K-Ras protein; Kinases; Lung cancer; Medical imaging; Medical research; Metabolism; Mice, Nude; Mutant Proteins - metabolism; Mutation; Nuclear transfer; Phenotype; Polymerase Chain Reaction; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins p21(ras); ras Proteins - metabolism; Recombination, Genetic - genetics; Rodents; Skin; Somatic cell nuclear transfer; Stem cells; Studies; Suidae; Thymidine; Thymidine kinase; Thymidine Kinase - metabolism; Transformation; Veterinary Science; Viability
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0090059

Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.