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Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog.
- Source :
-
Theriogenology [Theriogenology] 2020 Jul 01; Vol. 150, pp. 347-352. Date of Electronic Publication: 2020 Feb 19. - Publication Year :
- 2020
-
Abstract
- Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.<br /> (Copyright © 2020. Published by Elsevier Inc.)
- Subjects :
- Animals
CRISPR-Cas Systems
DNA End-Joining Repair physiology
Embryo Transfer
Fibroblast Growth Factor 5 genetics
Fibroblast Growth Factor 5 metabolism
Gene Editing methods
Gene Expression Regulation
Genotype
Keratins, Hair-Specific genetics
Keratins, Hair-Specific metabolism
Time Factors
Dogs physiology
Fertilization in Vitro veterinary
Gene Editing veterinary
In Vitro Oocyte Maturation Techniques veterinary
Oocytes physiology
Zygote physiology
Subjects
Details
- Language :
- English
- ISSN :
- 1879-3231
- Volume :
- 150
- Database :
- MEDLINE
- Journal :
- Theriogenology
- Publication Type :
- Academic Journal
- Accession number :
- 32088047
- Full Text :
- https://doi.org/10.1016/j.theriogenology.2020.02.003