1a. Objectives (from AD-416):
In this application, we provide a novel strategy to create disease resistant animals utilizing animal stem cells and cellular adaptive resistance (CAR) technology. This will produce animals with natural resistance to specific diseases resulting in animals that need less veterinary care and will significantly reduce livestock mortalities. The use of stem cells provides several advantages in producing livestock because stem cells are a highly scalable pluripotent cell source that provide sufficient number of cells for selection. The milestones will involve: 1) creating chicken stem cells; 2) developing cellular adaptive resistant chicken stem cells; 3) generate cell lines chimeric chicken offspring using CAR stems cells; 4) produce Newcastle disease virus (NDV) and avian influenza (AI) resistant birds using a conventional breeding program; and 5) produce birds resistant to NDV and AIV using standard breeding practices.
1b. Approach (from AD-416):
-Milestone 1: Creating chicken stem cells (4-8 months). Stem cell lines from 10 individuals will be created by the expression of pluripotent factors in adult (somatic) cells. This milestone will have been reached when cells possess stem cell morphology including large nucleoli, a high nuclear to cytoplasmic ratio, and colonial expansion. Cells will show increased telomerase activity relative to none stem cells and be capable of continual expansion without loss of stem cell character. -Milestone 2: Developing cellular adaptive resistant (CAR) chicken stem cells (9-12 months). CAR cells with resistance to Newcastle disease virus (NDV) and avian influenza (AI) will be developed by exposing these cells to multiple rounds of NDV and AI selection as both diseases cause cell death in culture. The success of this aim will be known when CAR cells that are not killed when exposed to these viruses are expanded creating viable cell lines. NDV and AI cells will undergo genetic and epigenetic comparisons to lines that are not resistant to identify key changes that result in resistance. -Milestone 3: Generate germ line chimeric chicken offspring using CAR stem cells (9-18 months). NDV and AI resistant stem cells will be injected into the vasculature of embryonic chicks resulting in the colonization of the gonad by these cells producing chimeric chickens. Stem cells and cells that have been altered will be tested for their ability to migrate to the gonad and produce juvenile and adult chimeric birds by detection of specific resistant and/or marker proteins and genes. - Milestone 4: Produce NDV and AI resistant birds using a conventional breeding program (18-24 months). To reach this milestone, the 20 male chimeric chickens containing disease resistant gametes produced in milestone 3 will be breed to 10 female chickens each (a total of 200 female chickens). The eggs will be collected from female chickens and allowed to hatch. F1 chicks carrying disease resistant cells will be identifiable by feather color with confirmation by genotype analysis. F1 chicks will be challenged by exposure to lethal levels of virus and assessed for survivability. F1 chicks found to be disease resistant will be breed for 5 generations with offspring at each stage being tested for maintained resistance. -Milestone 5: Produce native birds resistant to NDV and AI using standard breeding practices found in target communities.
3. Progress Report:
This research is directly related to inhouse objective 2.A - Define host response pathways modulated by Newcastle disease viral infections. The objectives of this research are partially meet. Two chicken derived pluripotent stem cell lines that resist infection with Newcastle Disease virus LaSota vaccine have been created. Primordial germ line cells and induced pluripotent cells are being tested for the capacity to differentiate in chicken embryos. These cells lines are being further evaluated for use to generate animals. Cell lines are also being evaluated for the capacity to resist infection with virulent Newcastle Disease viruses.