The experimental procedures and methods used in this study were approved by the Animal Welfare and Ethics Office (2019012A-CNU-174), Chungnam National University, Daejeon, and performed according to “The Guide for the Care and Use of Laboratory Animals” published by IACUC of Chungnam National University. Female mixed dogs from 2 to 6 years of age were used in this study as oocyte donors and embryo transfer recipients. The dogs were housed indoors and fed once daily with water ad libitum. All methods and protocols were performed in accordance with relevant guidelines and regulations.
Construction of prime editor vector and production of lentiviral particles.
The vector for PE was purchased from Addgene (Watertown, MA, USA : #135955) and modified to correct HD-related SNPs. Briefly, the CMV promoter was obtained by PCR using the primer sets 5'-gaattcttgacattgattattgactag-3' and 5'-tctagaaatttcgataagccagtaagc-3', and inserted into the vector by EcoRI and XbaI enzyme cuts. The pegRNA targeting the HD locus was newly synthesized and then added to the vector using PacⅠ and EcoRI. Finally, the vector was confirmed through sequencing. The lentiviral particles of PE vector were produced by commercial vendor (Lugen SCI, Inc., Bucheon, South Korea).
Collection and establishment of canine fibroblast cell lines, transduction, and transgene analysis.
Fibroblasts were collected from the ears of an 18-month-old Labrador retriever diagnosed with HD (donor patient). The primary fibroblasts were cultured in vitro using culture medium composed of DMEM-GlutaMAX, 15% fetal bovine serum, and 1% penicillin/streptomycin solution (GIBCO, Inc.). For transduction, 100 multiplicity of infection (MOI) of the PE lentiviral particles, containing 1 µg/mL of polybrene, was transduced into 1 × 105 fibroblasts per a well of 12-well plate. Transgene expression was confirmed by EGFP and integration of the vector was confirmed by sequence analysis.
SCNT and embryo transfer.
For generating gene corrected dogs, SCNT followed by embryo transfer was performed following the method described elsewhere 27. Briefly, in vivo matured oocytes with the first polar body were used for micromanipulation. Metaphase chromosomes were removed by aspiration from the oocytes. A single cell (C>T cell) was transferred into the perivitelline space of an enucleated oocyte, and each donor cell-cytoplast couplets were fused by two pulses of direct current (24-26 V for 15 µsec) using an Electro-Cell fusion apparatus. The fused SCNT embryos were chemical activated by incubating with 10 µM calcium ionophore (Sigma) and then 1.9 mM 6-dimethylaminopurine (6-DMAP). The activated SCNT embryos were surgically transferred into the oviducts of oestrus-synchronized surrogates. Pregnancy was confirmed by ultrasonography at 30 days after embryo transfer.
PCR validation and sequencing analysis.
Transgene integration into the genome of transduced fibroblasts and gene-corrected dogs was confirmed by PCR. The PCR primers used to validate the Cas9 sequence in the vector were 5'-catcgctattaccatggtgat-3' and 5'-ctcttgcagatagcagatcc-3'. These primer sets detected the linkage between the CMV promoter and dCas9 of the vector used in this study. Sequencing of the target locus was performed to validate the PE-mediated gene correction. The sequencing primers used were 5'-gacgccaagggagcagatatt-3' and 5'-cctctcttatgagaacagcat-3'.