Project Number: 1245-31000-103-03
Project Type: Specific Cooperative Agreement

Start Date: May 01, 2010
End Date: Apr 30, 2013

Objective:
To create bovine iPSCs for dairy cattle. The long term goal is to use iPSCs to improve the efficiency of cattle cloning. To provide a permanent source of a “clonable cell type”, and to create genetically modified cattle including gene targeting through site-directed genetic modifications.

Approach:
Pluripotency is the capacity of a single cell to generate in a flexible manner, all cell lineages of the developing and adult organism. Pluripotency is generated naturally during mammalian development through formation of the epiblast, the founder tissue of the embryo proper. Pluripotency can be regenerated outside of the embryo by reprogramming somatic cells. The elucidation of genes that control these stem-cell-like qualities has led to the development of methods in human, mouse, rat, and pig to alter gene expression of fibroblasts [or other differentiated somatic cells] to create iPSCs. This project will first explore the technology and identify the essential combination of genes, the gene delivery system, and culture conditions necessary to produce iPSCs in the bovine. Once the essential factors and conditions are demonstrated, the focus will shift to verifying the expression of known stem-cell markers and optimizing media and culture conditions for the long-term propagation and maintenance of the bovine iPSCs in vitro. Candidate bovine iPSCs will then be tested for their ability to differentiate into the three primary germ layers (endoderm, ectoderm and mesoderm) through the creation of teratomas in immune deficient SCID mice. Final proof that bovine iPSCs have been produced will be the demonstration that bovine iPSCs can be incorporated into virtually all tissues of calves as a result of the incorporation of these cells into the early embryo and their differentiation during organ formation. Once the pluripotency of our bovine iPSCs are verified with live progeny, the effort will shift to demonstrate our ability to create genetically modified cattle through the use of genetically modified iPSCs and standard embryo injection procedures.