The ability to efficiently modify the swine genome to generate genetically modified animals has the potential to provide a novel source of cells/tissues for clinical applications. Here we propose further development of the technology and its application to the development of genetically modified swine capable of high-level engraftment with human cells, and the testing and development of artificial organs. Specifically, we propose:
Aim 1. To develop a genetically modified pig deficient in B cells, T cells, and NK cells (we will refer to this model as pSCID for porcine Severe Combined Immunodeficient from now on).
Aim 1 a. will accomplish this by ablating B cells and T cells using the toxin DTA.
Aim 1 b will utilize a responder line carrying a conditional DTA activated by Cre expression and an inducer line expressing Cre under the control of lymphoid specific promoters. Both lines can be easily propagated, and distributed, and the pSCID phenotype induced when the two lines are crossed.
Aim2. To develop a pig hosting a human immune system.
Aim 2 a. We will utilize human/or swine bone marrow hematopoietic stem cells (HSC) to repopulate the immune system by injecting into a developing fetus prior to immune maturation. The porcine donors will be genetically labeled so that even in the absence of a pSCID we can complete this aim. Engraftment will be examined at one month, three months and six months of age by collection of bone marrow and peripheral blood.
Aim 2 b. Will examine the generated immune cells for the presence cell fusions between host and donor cells, presence of newly generated T cells using a T cell receptor excision circle assay (TREC), antibody receptor diversity by using oligonucleotide probes, proliferative responses using mixed leukocyte cultures, and examining novel T- cell specificities by immunizing with parvovirus, pseudorabies, and tetanus toxoid.
Aim 3. To develop a transgenic pig that will facilitate engraftment and proliferation of human hepatocytes.
Aim 3 a will focus on generation of a transgenic swine carrying Cre-ERT2, a tamoxifen inducible Cre, driven by a liver-specific promoter. Crossing this line with the inducible DTA line generated in Aim 1 b, will allow depletion of the porcine hepatocytes upon tamoxifen addition. This will allow transplanted non-transgenic human hepatocytes to gradually replace the porcine hepatocytes.
Aim 3 b will focus on analyzing the resulting hepatocytes at multiple levels including expression of mature hepatocyte markers, induction of drug metabolizing enzymes, liver cytoarchitecture and presence/absence of cell fusions.
This aim will allow us to determine the strengths and weaknesses of this xenotransplant organ regeneration approach so it can be used in the future to develop tissue and organs of greater complexity such as lung and cardiac tissue.
At completion of this proposal a number of unique transgenic swine will be available that can play a key role in the development and testing of artificial organs and tissue engineering but, in addition, have a broad range of uses in biomedical research. They can be used to study adoptive immunotherapy, normal and abnormal immune system development and function, study development and treatment for diseases such as leukemia, looking at disease progression and the development of vaccines for diseases such as AIDS, and study human tumor progression and treatment. In addition, the development of swine with livers largely composed of human hepatocytes will greatly facilitate pharmacological testing, the study of diseases such as hepatitis, development of models of liver-associated diseases such as cirrhosis, and eventual development of a human hepatocytes that can be transplanted back into humans.
|Koh, Sehwon; Piedrahita, Jorge A (2014) From "ES-like" cells to induced pluripotent stem cells: a historical perspective in domestic animals. Theriogenology 81:103-11|
|Lim, Ji-Hey; Piedrahita, Jorge A; Jackson, Lauren et al. (2010) Development of a model of sacrocaudal spinal cord injury in cloned Yucatan minipigs for cellular transplantation research. Cell Reprogram 12:689-97|
|Caballero, I; Piedrahita, J A (2009) Evaluation of the Serratia marcescens nuclease (NucA) as a transgenic cell ablation system in porcine. Anim Biotechnol 20:177-85|
|Bischoff, S R; Tsai, S; Hardison, N et al. (2009) Functional genomic approaches for the study of fetal/placental development in swine with special emphasis on imprinted genes. Soc Reprod Fertil Suppl 66:245-64|
|Zaunbrecher, Gretchen M; Dunne, Patrick W; Mir, Bashir et al. (2008) Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ). Anim Biotechnol 19:6-21|
|Lee, Eunsong; Estrada, Jose; Piedrahita, Jorge A (2008) A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods. Anim Biotechnol 19:71-9|
|Estrada, Jose L; Collins, Bruce; York, Abby et al. (2008) Successful cloning of the Yucatan minipig using commercial/occidental breeds as oocyte donors and embryo recipients. Cloning Stem Cells 10:287-96|
|Estrada, Jose; Sommer, Jeffrey; Collins, Bruce et al. (2007) Swine generated by somatic cell nuclear transfer have increased incidence of intrauterine growth restriction (IUGR). Cloning Stem Cells 9:229-36|
|Mir, Bashir; Zaunbrecher, Gretchen; Archer, Greg S et al. (2005) Progeny of somatic cell nuclear transfer (SCNT) pig clones are phenotypically similar to non-cloned pigs. Cloning Stem Cells 7:119-25|
|Dindot, Scott V; Farin, Peter W; Farin, Charlotte E et al. (2004) Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses. Biol Reprod 71:470-8|
Showing the most recent 10 out of 21 publications