Chemicals
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.
Animal Ethics
The all experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungnam National University (Approval No. : CNU-00012) and performed according to “The Guidelines for the Care and Use of Laboratory Animals” published by the IACUC of Chungnam National University. And this study was carried out in compliance with the ARRIVE guidelines. 14 female mongrel dogs between 2 and 6 years of age (body weight: 25–35 kg) were used as oocyte donors and surrogate mothers, and purchased from Honghwa Inc. (Nonsan, Korea). And 6 beagles from 2 to 6 years of age (body weight: 8–10 kg) purchased from ORIENT BIO Inc. (Seongnam, Korea) were used in this study for breeding. Dogs were raised indoors in separate cages with a 22–23°C temperature, 50–60% humidity and ventilation control system. They were fed commercial diet once daily and provided water ad libitum. They fed commercial diet (GOOD BOY JINDO, JEILpetfood, Seoul, Korea) at twice a day (09:00, 17:00), and freshwater was provided freely.
Construction of retroviral vector containing hDJ-1 and establishment of transgenic cell line
The gene sequence encoding the hDJ-1 protein was obtained from the human cDNA library. For N-terminal FLAG tagging, the hDJ-1 was cloned into pcDNA3.1(+) _Flag using hDJ-1 _Hind III_ forward (F) (5’-GGAAGCTTATGGCTTCCAAAAGAGCTCT-3’) and hDJ-1_Xho I_reverse (R) (5’- GGGCTCGAGCTAGTCTTTAAGAACAAGTG-3’) primers. The retroviral plasmid MSCV (pMSCV)-puro vector, which includes the MSCV promoter, long terminal repeat (LTR), virus packaging signal (Ψ+), and puromycin resistance gene, was used for hDJ-1 transfer (Fig. 7). After PCR amplification of hDJ-1, it was inserted into the pMSCV-puro vector using FLAG_Xho I_F (5’-CCCCTCGAGATGGACTACAAAGACGAT-3’) and hDJ-1_EcoRI_R (5’-GGGAATTCCTAGTCTTTAAGAACAAGTG-3’) primers.
The 293T cell line, which is derivative of human embryonic kidney 293 cells, were used as retrovirus-packaging cells, and polyethyleneimine (PEI) was used to transfect the cells with the FLAG-tagged hDJ-1 in the pMSCV vector. The retroviral vector-containing medium was harvested after 48 h of transfection. Thereafter, 106 CFFs/mL were seeded on a 100 mm dish (Falcon®, CORNING INC., Arizona, USA) and transfected with the retroviral vector in the presence of 6 µg/mL polybrene. The transfected cells were selected using puromycin. Finally, the live cells were isolated, proliferated, and stored at -196°C until further use.
Collection of in vivo matured oocytes
Blood was collected every day from 14 dogs on the first day of estrus, and serum progesterone (P4) concentrations were measured using a DSL-3900ACTIVE progesterone coated-tube radioimmunoassay kit (Diagnostic Systems Laboratories, Inc., TX, USA). The day when P4 concentration reached 4.0–7.0 ng/mL was considered as the day of ovulation. In vivo matured oocytes were collected by laparotomy after 3 d of ovulation. The dogs were anesthetized using 6 mg/kg ketamine and xylazine, and the anesthesia was maintained with 2% isoflurane. The ovaries and uterus were exteriorized by an aseptic surgical procedure. For oviductal flushing, an inverted flanged bulb steel needle (18-gauge, 7.5 cm) was inserted through the bursal slit and held in position using a plastic tube and hemostatic forcep. Subsequently, a 24-gauge intravenous (IV) catheter (Angiocath™ Plus, Becton Dickinson Korea Inc., Seoul, Korea) was inserted into the oviductal lumen near the uterotubal junction. Approximately 10 mL of media 199 (Gibco™, Thermo Fisher Scientific Inc., Massachusetts, USA) supplemented with 10 mM HEPES, 2 mM NaHCO3, 0.013 mM kanamycin, and 0.5% bovine serum albumin (BSA; Gibco™) was injected through the IV catheter into the oviduct. The 93 ovulated oocytes were flushed out via the steel needle and immediately transported to the laboratory.
Scnt And Embryo Transfer
The SCNT was performed according to a previously described protocol 28,29. First, the cumulus cells were removed from the in vivo-matured oocytes using 0.1% hyaluronidase. Thereafter, the first polar body and nucleus were removed in HEPES-buffered media 199 containing 5 µg/mL Hoechst 33342 and 5 µg/mL cytochalasin B using manipulators under an inverted microscope equipped with epifluorescence. Subsequently, a TG cell was injected into the perivitelline space of the enucleated oocyte. The couplets were electrically fused by two pulses of direct current (70–73 V for 15 µs) into a 0.26 M mannitol fusion medium containing 0.1 mM HEPES, 0.5 mM MgSO4, and 0.05% BSA using an Electro-Cell Fusion apparatus (NEPA Gene Co., Chiba, Japan). After incubation for 1 h 30 min in porcine zygote medium-3 (PZM-3), the embryos were examined. The fused embryos were chemically activated in 10 µM calcium ionophore for 4 min, washed in PZM, and incubated in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 3 h 30 min. Within 4 h of SCNT, the reconstructed embryos were transferred into the oviducts of naturally synchronized surrogate mother dogs using laparotomy. The cloned embryos were placed in the uterotubal junctions of the oviducts using an Argyle™ sterile tom-cat catheter (Medtronic Inc., Minnesota, USA). Pregnancy was confirmed using a MyLab30vetGold (Esaote, Genoa, Italy) ultrasound scanner with a variable-band convex array probe 25 to 30 d after embryo transfer.
Western Blotting
For western blot analysis, the CFFs, hDJ-1 transfected cells, and blood collected from control dogs as well as the hDJ-1 TG dog were lysed in radioimmunoprecipitation assay buffer [20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM ethylenediamine tetracetic acid (EDTA); 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA); 1 mM Na3VO4; 50 mM β-glycerophosphate; 50 mM NaF; 1% Triton X-100; and protease inhibitor cocktail]. The cell lysates were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. The nitrocellulose membranes were blocked for 2 h in 5% skim milk and incubated overnight at 4°C with anti-actin and anti-PARK7/DJ-1 (Abcam®, Cambridge, UK) antibodies. Thereafter, the membranes were washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, the membranes were developed, and the hDJ-1 proteins were visualized using LAS-4000 (Fujifilm Life Sciences, Massachusetts, USA).
Behavioral Tests Of The Tg Dog
When the TG dog appeared depressed, he was subjected to the knuckling test and 10 m walking test. The knuckling test, which determines the time taken by the dog to return his paw to the normal position after being flipped, is used to check the nerve reflex of the dog. In the 10 m walking test, the TG dog and control dogs were placed at a starting line. The tester, who was 10 m away from the starting line, enticed dogs to run, and the scorer recorded the time taken by each dog to reach the finish line.
Inverse Pcr
Genomic DNA of TG dogs was digested using BamH I and self-ligated using T4 ligase. Subsequently, PCR was conducted according to the following conditions: 95°C for 5 min; 35 cycles of 95°C for 30 s, 61.4°C for 40 s, and 72°C for 6 min; and a final extension at 72°C for 10 min. The first PCR was performed using IN_hDJ-1_F (5’-CTGAGAATCGTGTGGAAAAAGACGGCC-3’) and IN_hDJ-1 _R (5’-GATGACCAGTCTTTTGGAAGCCAT-3’) primers. The second PCR was performed using the PCR products of the first PCR. For this, IN_hDJ-1_F (5’-CTGAGAATCGTGTGGAAAAAGACGGCC-3’) and IN_LTR _R (5’-GCCCATATTCTGCTGTCTCTCTGTTCC-3’) primers were used, and the insertion sites of hDJ-1 were identified by sequencing.
Surgical Insemination Of Dogs
To confirm the germ-line transmission of hDJ-1, the TG dog was artificially bred with wild-type female dogs via surgical insemination. Semen was collected from the TG dog and washed with phosphate buffered saline (PBS; Gibco™). The sperms were diluted with 1 mL PBS and stored at 37°C until artificial insemination (AI) was performed on the bitches via laparotomy. After 2 d of ovulation, the uteri of the 5 female dogs were exposed through abdominal incisions. Sperm samples were injected into the uterine horn using a 3 mL syringe and a 24-gauge IV catheter. Approximately 28 d after AI, pregnancy was confirmed using ultrasound imaging, and after 60 d of AI, the offspring of hDJ-1 TG dog were born via vaginal delivery.
In vivo MRI
The hDJ-1 TG dog and one wild-type control dog were subjected in vivo MRI of the brain to evaluate the structural changes. The TG dog was 3-y-old and control dog was 2-y-old, and weighed 9–10 kg. They were fasted for at least 20 h prior to the scan. Before conducting the MRI, each dog was anesthetized with ketamine (6 mg/kg, IV). Subsequently, the dog was transferred to the MRI table and immobilized in the prone position. The anesthesia was maintained with 2% isoflurane in 99.9% oxygen (2 L/min) throughout the procedure. Pulse rate and body temperature of the dogs were monitored continuously, and the body temperature was maintained with a warm blanket surrounding the animal. All MRI experiments were performed using a Philips 3T Achieva scanner (Philips Medical System, Best, Netherlands) with a 32-channel head coil at the Korea National Primate Center 30. The MRI included whole-head T1-weighted and T2-weighted images. The T1-weighted imaging was performed with a three-dimensional (3D) turbo field echo (TFE) sequence with the following parameters: repetition time (TR) = 14 ms, echo time (TE) = 6.8 ms, field of view (FOV) = 13 × 13 cm, 150 slices, slice thickness = 0.5 mm, acquisition matrix size = 260 × 165, voxel size = 0.5 x 0.79 x 0.5 mm, 4 number of signals averaged (NSA). The 3D T2-weighted imaging was performed with a 3D turbo spin echo (TSE) sequence with the following parameters: TR = 2,500 ms, TE = 217 ms, FOV = 13 × 13 cm, 150 slices, slice thickness = 0.5 mm, acquisition matrix size = 260 × 260, voxel size = 0.5 x 0.5 x 0.5 mm, 1 NSA.
In vivo PET-CT imaging
All in vivo PET images were acquired using a PET-CT scanner (Biograph-mCT s20-3R; Siemens Medical Systems, Knoxville, USA). After the MRI, the dogs were transferred to the PET-CT table and immobilized in the prone position. The anesthesia was maintained with 2% sevoflurane in 99.9% oxygen (2 L/min). The [18F]-FP-CIT (DuChemBio Co., Ltd., Daejeon, Korea) PET imaging was performed to detect the dopamine transporters 31. The dogs were administered 5 mCi of [18F]-FP-CIT intravenously, followed by CT scan for attenuation correction. Scanning began at the same time as radiotracer injection. The brain PET images of 400 × 400 matrix size were acquired by a 39-frame dynamic sequence for 120 min, according to the following pattern: 15 sec × 8, 30 sec × 6, 60 sec × 5, 120 sec × 5, 300 sec × 10, and 600 sec × 5. The images were reconstructed using a standard iterative algorithm (OSEM 3D + TOF) and post-processed using Syngo VG51C software (Siemens Medical Solutions, Erlangen, Germany) to visualize multiplanar fusion images.
Pet/mri Fusion Imaging And Quantitative Analysis
To fuse the images obtained during PET and MRI, the MRI data were processed using the software supplied by the manufacturer and converted to the DICOM format for co-registration with PET images. The PET and MR images were co-registered on IRW software (Inveon Research Workplace, Siemens Medical Solutions, USA) by rigid and manual registration 32. The striatal binding potential was calculated by Logan’s non-invasive graphical method using a reference region 33. The striatum was segmented by threshold-based automatic delineation of volume of interest (VOI) with the aid of MRI. The VOI of the cerebellum was selected as the reference region.
Statistical analysis
For each experiment there were at least 3 replicates, and data were analyzed using SPSS statistics 24 software (IBM SPPS statistics, Chicago, IL, USA).