Project Number: 8042-31440-001-05-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2015
End Date: Jul 14, 2019
Selective breeding in domestic farm animals has been very successful and resulted in a significant enhancement of specific production traits in farm animals, but along with preferred traits has also been the co-segregation of deleterious traits. The ability to selectively remove undesirable production traits or introduce beneficial traits is of even greater interest because unprecedented challenges such as climate change, decreases in farmland, movement of humans and animals that increases exposure to novel disease, and projected increases in meat consumption are occurring. A primary goal of the laboratory is to develop and refine tools for gene engineering of farm animals to improve their production traits. An alternative to the direct editing of a gene(s) of interest in an embryo is the editing of the gene(s) within a cultured cell whose nucleus will then be used for nuclear transfer, i.e., animal cloning. This approach allows for the gene-edited cell to be expanded into a cell line which can then be verified as having the correct genetic change before embryo generation. Also, the cell line can be maintained indefinitely. However, nuclear transfer with a somatic cell means that the cell’s nucleus must be efficiently reprogrammed to a state of “pluripotency” (i.e., can give rise to all three germ layers) to yield the induced pluripotent stem cell (iPSC). It is thought that the use of the pluripotent iPSC rather than somatic cell would be advantageous because the pluripotent stem cell’s chromatin is more “zygote-like” and thus more easily reprogrammed. Being able to combine gene editing and nuclear transfer along with gender selection would be of great value where the genes targeted are of consequence only in the male or the female, but not in both. The establishment of transcriptome and methylome data sets of bovine in vivo embryos will provide additional and necessary information for determining what gene modifications can be successfully pursued in cattle in the context of early embryo development and, i.e., genetic changes, that will not perturb normal development. The major goals will be to: a) Generate characterized, bovine iPSCs for use as donor nuclei to improve nuclear transfer. b) Identify sperm surface proteins that will allow highly efficient, reproducible separation of bovine X- and Y-sperm for gene-editing along with nuclear transfer or for cloning where gender specificity is essential.
Historically, ABBL has had success with creating genetically modified farm animals with pronuclear microinjection and somatic cell transfer technology and we are currently developing gene editing technology. To date, iPSC that are equivalent to mouse embryonic stem cells (ESC) have not been generated for the bovine. Work with rodents has shown that both “naive” and “primed” iPSC states exist and the naive state has been shown to possess pluripotency, i.e., are equivalent to mouse ESC. The availability of naïve state iPSCs would allow for a continuous supply of pluripotent cells that would be expect to improve nuclear cloning efficiency and thus the efficiency of genetic engineering to improve production and health of cattle. In addition, an improved method for X-/Y-sperm sorting would have application to enhance SCNT and other gene editing technologies where gender specificity would be beneficial. Sub-objective 1: a. Naive bovine iPSC will be generated by University of Connecticut (UCONN) scientists through the reprogramming of bovine XX (female) cells by utilizing select subsets of reprogramming factors and by optimizing culture conditions. b. Once the essential factors and conditions are demonstrated for bovine iPSC creation, the focus will shift to verifying the expression of known naive stem-cell markers in the iPSC and to optimizing culture conditions for the long-term propagation and maintenance of the bovine iPSCs in vitro. c. Candidate bovine iPSCs will be tested by UCONN and ARS scientist to examine their phenotype by assessing their expression of pluripotency markers, their ability to differentiate into the three primary germ layers and their propensity to form teratomas in immunodeficient mice. d. The long-term goal is to compare the efficiencies of nuclear transfer as well as the normality of cloned embryos and offspring resulting from naive bovine iPSCs vs. differentiated somatic cells (UCONN and ARS). Sub-objective 2: a. The UCONN scientist will extract bovine sperm protein and prepare samples for mass spectrometry analyses. b. The tandem mass spectrometry analysis and subsequent bioinformatic analysis will be performed at ARS. c. The long-term goal is to edit the bovine genome on differentially expressed proteins on the sperm heads of X- and Y-bearing sperm. Such genome-edited cells will then be used for nuclear transfer to improve the efficiency of bovine sperm sorting (UCONN). Sub-objective 3: a. In our prior work, transcription profiling of in vivo bovine embryos were established using RNA-seq. Analysis of developmental pathways, specific groups of functional genes and novel gene expression will be conducted (UCONN). Comparisons to other ungulate species as well as to embryos produced by in vitro fertilization will be performed (UCONN). b. New in vivo embryos will be collected by UCONN’s collaborator, Dr. Jingbo Chen, and methylomic (UCONN) and data analyses correlating methylations with gene expression will be conducted (UCONN and ARS).