Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
In the sheep industry, tail docking, or the amputation of the tail, is a common practice used to prevent illness and injury. While beneficial in improving rear hygiene for the animals for the prevention of fly-borne illness, the manual removal of the tails is a painful procedure with the potential to cause other health issues later in life. The goal of this project is to provide an alternative to tail docking for the improvement of animal welfare in this production system. To achieve this, a genetic method of reducing tail length will be devised. The development of this genetic docking strategy will be applicable to long-tailed sheep breeds worldwide.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
0%
Developmental
50%
Goals / Objectives
The primary goal of this project is to develop a strategy for the generation of short-tailed sheep through manipulation of the T-box transcription factor T (TBXT) gene. This will be accomplished through the following objectives. To ascertain the efficiency of engineering ovine embryos with a missense mutation in exon 2 of TBXT via CRISPR-Cas9 gene editing, we will 1) establish a pipeline for the in vitro production of ovine embryos and 2) determine the optimal conditions for knocking in the target mutation. For the generation of the edited sheep, we aim to 1) achieve full-term pregnancies through embryo transfer, 2) characterize the genotypes and phenotypes of live offspring produced, and 3) ensure normal growth and development in the gene-edited sheep.
Project Methods
The generation of short-tailed sheep will begin with the collection of cumulus-oocyte complexes (COCs). Donor sheep will be synchronized with reproductive hormones to superstimulate follicle development. Follicles will be aspirated within the body cavity using laparoscopic-assisted ovum pick-up (LOPU). The collected COCs will be matured for 24 hours before being fertilized with cryopreserved sperm. Following incubation with the sperm, the cumulus cells will be removed for electroporation to allow for the entry of CRISPR-Cas9 gene editing reagents. The edited embryos will then recover before being transferred into synchronized recipient sheep through laparoscopic-assisted embryo transfer. Leftover embryos will be lysed and genotyped through polymerase chain reaction (PCR) and subsequent sequencing to determine mutation efficiency. Determining the mutation efficiency of our gene editing strategy is the first milestone in this project with success being an efficiency of 50%.Thirty days after embryo transfer, pregnancies will be confirmed via ultrasound. Ewes that are not pregnant will be recycled into the recipient pool. About five months after fertilization, the ewes will give birth and the offspring will be genotyped using tissue from an ear punch or a blood sample. Body weights and tail lengths will be recorded for each lamb. X-ray visualization will be used to count vertebrae number. Throughout the first year of life, weight, height, girth, body length, and tail length will be measured monthly. At maturity, the short-tailed sheep will be bred through natural mating and reproductive soundness will be evaluated by pregnancy rate and prolificacy. The second-generation offspring will be evaluated for tail length, vertebrae number, and genotype at parturition. All TBXT-mutant sheep will be compared to their wildtype counterparts to detect differences in growth, development, reproductive fitness, and tail phenotype. Our milestone for this portion of the project is the production of at least 8 offspring with reduced tail lengths and vertebrae number.