1a. Objectives (from AD-416)
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.
1b. Approach (from AD-416)
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.
3. Progress Report
This work supports the National Program 101 mission statement in the area of developing information, tools, and technologies that can be used to improve animal production systems. Significant progress was made on Component 1: Understanding, improving, and effectively using animal genetic and genomic resources. Progress on this project focuses on Problem 1A, the need for developing genome-enabling tools and reagents for livestock (pig and cattle). Progress was made in the development of specialized cell lines from food animals that can be used to study gene expression and developing improved techniques and methodology for transfer of genes within and across species. There are currently no embryonic stem cell lines for livestock species with this ability. We are engaged in producing induced Pluripotent Stem Cells (iPSCs) for cattle, pig and goat breeds. iPSCs constitute an emerging technology for creating cell lines that mimic embryonic stem cells with the advantage that they can be maintained in culture long term, allowing genetic manipulations such as gene targeting. These tools will not only be useful for traditional animal production research applications (reproduction, growth and development, nutrient intake and utilization, product quality), but will also be used to decrease the environmental footprint of animal production, improve animal health, well-being and resistance to disease, and enhance food safety. There has not been significant advancement of either common goal of ARS and U Conn scientists to first develop bovine induced pluripotent stem cells with foreign genetic material added to the genome and, second, to generate these cells lacking any foreign genetic material in the genome for the purpose of performing hypothesis driven research in the study of bovine functional genomics. The ARS and U Conn have both achieved reprogramming of murine fibroblasts. Success on this project will allow for rapid creation of bovine strains with enhanced production traits. Activity on this agreement is monitored through conference calls and site visits.