Supplementary Information
Care and Use of Animals
In this study, mixed breed female dogsfrom 1 to 3 years of age were used. The dogs were cared for in facilities, and using procedures, which exceeded the standards established by the Seoul National University for Accreditation of Laboratory Animal Care. The study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals in Seoul National University.
Chemicals
Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Vaginal Cytology and Progesterone levels
Females were examined on a daily basis for vulva swelling and serosanguinous discharge from the starting day of natural heat. Smears were obtained daily from the day of initial sign of proestrus to the surgery for oocyte retrieval. Smears were collected by inserting a swab into the lips of the vulva, then rolling it on a glass slide and staining with a Diff-Quik® staining.In order to measure serum progesterone concentration, blood (3 to 5ml) was collected every day and centrifuged. Sera were analyzed using a DSL-3900 ACTIVE® Progesterone Coated-Tube Radioimmunoassay Kit (Diagnostic Systems Laboratories, Inc., TX).The day on which the progesterone concentration initially reached 4.0 to 7.5ng/ml was regarded as the day of ovulation as described by Hase et al1.
Donor cell preparation for somatic cell nuclear transfer
Adult fibroblasts were isolated from an ear skin biopsy of a three-year oldmale Afghan Hound. Small pieces of ear tissue fragment were washed three times in DPBS and minced with a surgical blade. The minced tissue was dissociated in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Rockville, MD) supplemented with 0.25% (w/v) trypsin and 1 mM EDTA (Life Technologies) for 1 hour at 37oC. Trypsinized cells were washed once in Ca2+- and Mg2+-free DPBS by centrifugation at 300 x g for 2 min, and seeded into 100-mm plastic culture dishes (Becton Dickinson, Lincoln Park, NJ). Seeded cells were subsequently cultured for 6 to 8 days in DMEM supplemented with 10% (v/v) FBS (Life Technologies), 1 mM glutamine (Life Technologies), 25 mM NaHCO3 and 1% (v/v) minimal essential medium (MEM) nonessential amino acid solution (Life Technologies) at 39C in a humidified atmosphere of 5% CO2 and 95% air. After removal of unattached clumps of cells or explants, attached cells were further cultured to confluency. These cells were subcultured at intervals of 4 to 6 days by trypsinization for 1 min using 0.1% trypsin/0.02% EDTA, allocated to three new dishes for further passaging and then stored in freezing medium in liquid nitrogen at –196 C using freezing medium. The freezing medium consisted of 80% (v/v) DMEM, 10% (v/v) DMSO and 10% (v/v) FBS. Cells at passages 2 to 5 were used for somatic cell nuclear transfer (SCNT). Prior to SCNT, cells were thawed, cultured for 3 to 4 days until 100% confluent, and retrieved from the monolayer by trypsinization for 1 min.
Flushing of in vivo canine oocytes
Oocytes were retrieved from anesthetized females by laparotomy. The fimbriated end of the oviduct was accessed through the bursal slit and cannulated using an inverted flanged bulb needle. The needle was held in place by a surgical ligature, which was tied using a quick-release device using 3 cm plastic tube and hemostatic forceps. The base of the oviduct, just above the uterotubal junction, was visualized using digital pressure to blanch the surrounding tissue and the uterine tube lumen, and cannulated using a fine hypodermic needle(24 gauge) attached to a syringe filled with embryo collection medium, Hepes-buffered tissue culture medium (TCM)-199supplemented 10% (v/v) FBS, 2 mM NaHCO3, 5 mg/ml BSA (Life Technologies). Collected in vivo matured oocytes were transported into the laboratory within 5 minutes in Hepes-buffered TCM-199 at 38.5oC.
Preparation of Recipient Oocytes for Somatic Cell Nuclear Transfer
Cumulus cells from the in vivo matured oocytes at metaphase II were removed by repeated pipetting in 0.1% (v/v) hyaluronidase (from bovine testis) in Hepes-buffered Ca2+-free CR2 medium2 with amino acids (hCR2aa). Oocytes were then stained with 5 g/mL bisbenzimide (Hoechst 33342) for 5 min and observed under an inverted microscope equipped with epifluorescence at 200x magnification. Each oocytewas held with a holding micropipette (150 m inner diameter), then enucleated with a micromanipulator (Nikon-Narishige, Tokyo, Japan) in hCR2aa supplemented with 10% (v/v) FBS and 5 g/mL cytochalasin B.The first polar body and adjacent cytoplasm, containing the metaphase-II chromosomes, were removed using an aspiration pipette. The enucleated oocytes were placed in TCM-199 supplemented with 10% (v/v) FBS and used for SCNT.
Microinjection, Fusion, Activation and Embryo Culture
A single canine ear fibroblast was deposited into the perivitelline space of an enucleated oocyte treated with 100 μg/mL phytohemagglutinin in hCR2aa to improve the incorporation of donor somatic cell with recipient cytoplast. The couplets were subsequently placed in a fusion medium comprising 0.26 M mannitol, 0.1 mM MgSO4, 0.5 mM Hepes and 0.05% (w/v) BSA, and transferred into a cell fusion chamber with a stainless steel wire electrode (BTX 453, 3.2 mm gap; BTX, San Diego, CA). After equilibration for 3 min,couplets received two DC pulses of 3.0 ~ 3.5 kV/cm voltages for 15 sec using a BTX Electro-cell Manipulator.The fusion of the donor cell and the ooplast was observed 1 hour after electric stimulation under a stereomicroscope. Only fused embryos were selected and cultured for 3 hours in modified synthetic oviductal fluid (mSOF) as previously described3.The osmolarity and pH of mSOF were 270 to 280 mOsm and 7.2 to 7.3, respectively.Chemical activation of reconstructed canine embryos was induced by incubating embryos in mSOF containing 10 μM ionophore for 4 min at 39C. Reconstructed embryos were then washed and further incubated for 4 hours in mSOF supplemented with 1.9 mM of 6-dimethylaminopurine. After reconstruction, a group of 5 to 6 embryos was cultured in 25 μL microdrops of mSOF overlaid with mineral oil before embryo transfer.
Embryo Transfer and Pregnancy Diagnosis
The reconstructed embryos (5 to 12 embryos/recipient dog) were surgically transferred into the oviduct or the uterus of the surrogate mother depending on their developmental stage;the fused couplets within 4hours after reconstruction and in vitro cultured 2- or 4-cell embryos were transferred into the oviduct, while > 8-cell embryos were transferred into the uterus. Recipients synchronized in natural estrus were used for surrogates. For surgical transfer, anesthesia was induced with 0.1mg/kg acepromazine and 6mg/kg propofol, and general anesthesia was maintained with 2% isoflurane. While in dorsal recumbency, the recipients were aseptically prepared for surgery and a caudal ventral incision was made to expose the reproductive tract. Reconstructed embryos were placed in the ampullar by feeding a 3.5F Tom cat catheter (Sherwood, St.Louis, MO) through the oviduct. Pregnancies were detected using a SONOACE 9900 (Medison Co. LTD, Seoul, Korea) ultrasound scanner with an attached 7.0 MHZ linear probe 22 days post transfer. Pregnancy was monitored by ultrasound every 2 weeks after initial confirmation.
DNA Extraction and Microsatellite Analysis
Parentage analysis was performed in the donor dog, nuclear donor fibroblasts, cloned puppies and surrogate recipients to confirm the genetic identity (Table S1). Tissue fragments were obtained from the tail of the cloned puppies and blood samples were collected from the donor and surrogate mother. The tissue fragments, bloods, and trypsinized donor cells were incubated with a lysis buffer [0.05 M Tris (pH 8.0), 0.05 M EDTA (pH 8.0), 0.5% SDS] supplemented with 400 g proteinase K overnight, followed by phenol extraction and ethanol precipitation. The isolated genomic DNA samples were dissolved in 50 µl TE and used for microsatellite assay with eight canine specific markers4-7. Length variations were assayed by polymerase chain reaction (PCR) amplification with fluorescently labeled locus-specific primers and PAGE on an automated DNA sequencer (ABI 373: Applied Biosystems, Foster City, CA). Proprietary software (GeneScan and Genotyper; Applied Biosystems) was used to estimate PCR product size in nucleotides.
References
1. Hase. M. et al. J. Vet. Med. Sci. 62, 243-248 (2000).
2. Rosenkrans, C.F.J.et al.Biol. Reprod. 49, 459-462 (1993).
3. Jang, G. et al. Reprod. Fertil. Dev.15, 179-185 (2003).
4. Francisco, L.V. et al. Mamm. Genome7, 359-362 (1996).
5. Neff, M.W. et al. Genetics. 151, 803-820 (1999).
6. Richman, M. et al. J. Biochem. Biophys. Methods47, 137-149 (2001).
7. Denise, S. et al.Animal Genetics. 35, 14-17 (2004).
Corrected Table S1. Analysis of canine-specific microsatellite loci.
Canine markers / Donor-dog (Afghan hound) / Snuppy*(Tail-tissue fragment) / Surrogate female (Labrador retriever; blood leukocytes) / NT-2†
(Tail-tissue fragment) / Surrogate female (mongrel; blood leukocytes)
Blood leukocytes / Nuclear-donor fibroblasts
Peak 1 / Peak 2 / Peak 1 / Peak 2 / Peak 1 / Peak 2 / Peak 1 / Peak 2 / Peak 1 / Peak 2 / Peak 1 / Peak 2
PEZ01 / 110 / 118 / 110 / 118 / 110 / 118 / 118 / 110 / 118 / 119 / 123
PEZ02 / 122 / - / 122 / - / 122 / - / 114 / 126 / 122 / - / 126 / 134
PEZ15 / 214 / 214 / 214 / 208 / 217 / 214 / 214 / 244
REN162B09 / 191 / 195 / 191 / 195 / 191 / 195 / 181 / 191 / 195 / 182 / 192
REN105L03 / 239 / 239 / 239 / 239 / 248 / 239 / 249 / 252
REN165M10 / 187 / 191 / 187 / 191 / 187 / 191 / 177 / 187 / 187 / 191 / 179 / 187
FH2140 / 122 / 122 / 122 / 131 / 122 / 121 / 131
Seven‡ canine specific markers4 were used for microsatellite assay. PEZ markers are identified in US patent 05874217; markers for REN162B09, REN105L03, REN165M10, and FH2140 are listed at
J. Y. Han (Seoul National University) and S. J. Oh (NLRI) assisted with the microsatellite analysis.
*Snuppy was born on 24 April 2005; †NT-2 was born on 29 May 2005.
‡The PEZ08 marker has been removed from the Table.
Corrections are all in red.