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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #308426

Research Project: DEVELOPING GENETIC BIOTECHNOLOGIES FOR INCREASED FOOD ANIMAL PRODUCTION, INCLUDING NOVEL ANTIMICROBIALS FOR IMPROVED HEALTH & PRODUCT SAFETY

Location: Animal Biosciences & Biotechnology Laboratory

Title: Generation of induced Pluripotent Stem Cells from Domestic Goats - Capra hircus

Author
item SANDMAIER, SHELLEY - University Of Maryland
item NANDAL, ANJALI - University Of Maryland
item Powell, Anne
item Garrett, Wesley
item Blomberg, Le Ann
item Donovan, David
item Talbot, Neil
item TELUGU, BHANU PRAKASH - University Of Maryland

Submitted to: Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2015
Publication Date: 6/26/2015
Citation: Sandmaier, S.E., Nandal, A., Powell, A.M., Garrett, W.M., Blomberg, L., Donovan, D.M., Talbot, N.C., Telugu, B.V. 2015. Generation of induced Pluripotent Stem Cells from Domestic Goats - Capra hircus. Molecular Reproduction and Development. 82:709–721.

Interpretive Summary: Genetic modification of goats provides unique opportunities for improving goat health and products, and it also enables the production of biological pharmaceuticals in goat milk and the creation of goat models of human diseases. However, the genetic modification of goats has languished without significant technological gains for the past two decades due to the lack of authentic goat embryonic stem cell lines, the type of cells that revolutionized the genetic modification of mice. As an alternative to the long sought after goat ESC lines, we have generated goat induced pluripotent stem cell lines by artificially expressing so-called reprogramming factors in ordinary goat body cells. The resulting giPSC exhibit many characteristics of true ESC and, therefore, may be useful as a tool to improve the genetic engineering of goats.

Technical Abstract: The creation of genetically modified (GM) goats provides a powerful method for improving animal health, enhancing production traits, animal pharming, and ensuring food safety, all of which are high priority goals for animal agriculture. However, GM goats and the GM livestock field in general have languished without significant technological gains for the past two decades due to the lack of authentic goat embryonic stem cell (ESC) lines that revolutionized the GM mouse field. The availability of goat ESC lines that are characteristically immortal in culture would be of enormous benefit for creating GM animals. As an alternative to the long sought after goat ESC lines, we have generated goat induced pluripotent stem cell (giPSC) lines by forced expression of OCT4, SOX2, MYC, KLF4, LIN-28 and NANOG reprogramming factors (RF) in combination with a MIR302/367 cluster, all obtained from another ruminant, bovine, and delivered by cleavable (floxed) lentiviral vectors (LV). In order to minimize integrations, the RF genes were assembled with porcine teschovirus-1 2A (P2A)-self cleaving peptides that facilitate tri-cistronic expression from each lentiviral vector. Following transduction with the LV vectors, the cells were cultured in a semi-defined serum-free medium (KOSR) containing FGF and/or LIF on irradiated mouse feeder cells. The resulting giPSC exhibit cell and colony morphology typical of human ESC, i.e., well defined borders and a high nuclear to cytoplasmic ratio, a short cell-cycle interval, alkaline phosphatase expression, and the generation of teratomas in vivo. Additionally, the giPSC have demonstrated the ability to differentiate into directed lineages in vitro. These experiments constitute the necessary first steps in establishing integration and footprint free iPSC lines from goats and other ruminants.