Cell culture
Canis lupus familiaris canine fetal fibroblast cells (K9 Fetus 1), were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% Glutamax (Life Technologies), 1% minimum essential medium non-essential amino acids (Life Technologies), 1% antibiotic-antimycotic (Life Technologies), and 0.1% 2-mercaptoethanol (Life Technologies).
For the temozolomide (TMZ; Sigma Aldrich, St. Louis, MO, USA) treatment experiment, cells were harvested using Trypsin-EDTA, counted, and reseeded at a density of 1 × 106 cells per 100-mm culture plate. Twelve hours later, 100 nM of TMZ was added to each well.
Guide RNA design
To design gRNAs targeting the canine TP53 locus, all candidates with NGG-protospacer adjacent motif (PAM) sequences in all exons of the gene were identified using Feng Zhang laboratory’s Target finder program (https://zlab.bio/guide-design-resources). The program’s automatic off-target screening system was not available for dog species; therefore, we narrowed the results using the following steps. First, gRNAs near the N-terminus within the third exon of each gene were selected because their partial functional products could be produced if their functional domains were involved. Next, candidates with a lower binding potential compared to other genomic regions (gRNAs ≤ max score 30.2), particularly to coding sequences, were identified using the Basic Local Alignment Search Tool (BLAST) to avoid off-target alterations. Nineteen gRNAs for TP53 were selected using this process and the final three gRNAs were selected based on their lowest non-specific binding potential, in accordance with the Max score determined using the BLAST algorithm.
Vector construction
To construct a CRISPR/Cas9 plasmid vector targeting canine TP53, the U6-stuffer-hSpCas9 sequence was digested with Kpn1 (blunted by Klenow fragment) and BamH1 from lentiCRISPRv2 (#52961; Addgene, Cambridge, MA, USA) [37]. The fragment was inserted into the Nru1 and BamH1 sites of pcDNA3.1(+) (Thermo Fisher Scientific, Waltham, MA, USA). The enhanced green fluorescence protein (EGFP) sequence was digested with EcoR1 and Not1 from pEGFP-N2 (Clontech Laboratories Inc., Mountain View, CA, USA) and the synthesized E2A peptide sequence was inserted into the pcDNA3.1-CRISPR plasmid. Finally, the three designed gRNA sequences were synthesized and inserted into the two BsmB1 sites flanking the stuffer sequence.
Construction of canine TP53 KO fibroblasts
CRISPR/Cas9 vectors targeting TP53 were transfected transiently using Polyexpress™ (Excellgene, Rockville, MD, USA) into K9 fetus 1 cells at passage 1 according to the manufacturer’s instructions. The cells were then cultured and transferred into culture plates until untransfected cells showed senescence phenotypes. Finally, cell colonies with a normal cellular morphology and extended life span were separated and maintained independently.
Semi-solid agar culture assay
A 1.5 mL lower layer of 0.72% agar in DMEM containing 10% FBS was added to each well of a 6-well plate and allowed to solidify at room temperature (19–21 °C). Cells tested by the semi-solid agar assay were suspended in a plating layer (1.5 mL) of 0.28% agar in DMEM containing 10% FBS and added to the wells. The agar was allowed to solidify at room temperature and then incubated at 37 °C in a humidified CO2 incubator. To prevent the agar from drying, 500 μL of DMEM containing 10% FBS was added to each well every 2–3 days.
Cell growth counting
For cell proliferation analysis, 1.5 × 104 K9 fetus 1 control cells and TP53 KO cells were seeded into 6-well plates and cultured in the presence of 10% FBS for 6 days. Cells were harvested via trypsinization at various times and stained with Trypan blue. Viable cells were counted using a hematocytometer and inverted microscope. The number of cells was averaged over three independent experiments.
Western blotting
For western blot analysis, whole cell extracts were prepared in radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, and 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM NaF, 1 mM Na3VO4, and a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein levels were quantified using the Bradford assay reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA) according to standard protocols. Membranes were immunoblotted with antibodies against HRAS (Merck Millipore, Billerica, MA, USA), TP53, p21, SV40 T antigen, and β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA) in 3% bovine serum albumin in TBST. After primary antibody incubation, the membranes were washed and probed with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Pierce Biotechnology, Rockford, IL, USA). β-Actin was used as a loading control.
Canine TP53 target sequencing
Genomic DNA was extracted from canine fetal fibroblasts by using a Wizard Genomic DNA Extraction Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Next, exon 3 of TP53 in each cell line was amplified using Takara Ex taq™ (Takara Bio Inc., Shiga, Japan) and primer sets, forward 5′-CTGGTAAGGACTGGGTGTGG-3′ and reverse 5′-GCCACTGACCGTCCAAGTAA-3′under the following conditions in the thermal cycler: 60 s at 95 °C; 30 cycles of: 30 s at 95 °C, 40 s at 58 °C, and 60 s at 72 °C; followed by 10 min at 72 °C. Each product was ligated into a pGEM T-easy vector (Promega), according to the manufacturer’s instructions, and then sequenced using an ABI BigDye® terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA).
In vivo tumorigenesis assay
To establish subcutaneous xenograft models, 2 × 106 cells of each cell line in phosphate-buffered saline were mixed with 50% Matrigel (Invitrogen, Carlsbad, CA, USA) and transplanted subcutaneously into 5–6-week-old BALB/c nu/nu mice. The size of each tumor was calculated using the following formula: tumor volume (mm3) = longest diameter of tumor (mm) × shortest diameter of tumor (mm)2/2.
Karyotyping
Normal canine and TP53 KO fibroblasts were karyotyped commercially by GenDix, Inc. (Seoul, Korea). The results of karyotyping of the two fibroblast lines were analyzed using the ChIPS-Karyo program, a chromosome image processing system. Analysis data and all terms were represented by an International System for Human Cytogenomic Nomenclature 2016 (ISCN 2016).
Surveyor assay
Potential off-target genes of each gRNA were predicted using the BLAST algorithm and final target genes for the surveyor assay were selected in accordance with the matched number of nucleotides to each TP53 gRNA (≥10 of 20) and presence of a NGG PAM sequence.
Genomic DNA was extracted from canine fetal fibroblasts and TP53 KO cells using a Wizard Genomic DNA Extraction Kit (Promega) according to the manufacturer’s instructions. The surveyor assay was conducted using the Guide-it™ Mutation Detection Kit (Clontech Laboratories, Inc.) according to the manufacturer’s instructions. During the amplification step, PCR was performed using 100 ng of extracted canine genomic DNA. Primer sequences are listed in Additional file: Table S2.
Ovulation determination
Unless otherwise indicated, all reagents were obtained from Sigma-Aldrich. All donors and recipients employed in the study showed spontaneous estrous. The estrous stage was examined weekly by observing for vulval bleeding to detect the onset of the heat period. During heat, a 2 mL blood sample was collected daily by cephalic venipuncture and serum P4 levels in the blood samples were measured by electrochemiluminescence immunoassay (Cobas e411, Roche Diagnostics, Mannheim, Germany; intra- and inter-assay coefficients of variation < 4%). Ovarian ultrasonographies were periodically performed twice per day when serum P4 levels were found to be increased by more than 2 ng/mL. The time of ovulation was designated as the time when the ovaries became difficult to find for an apparent decrease in the number or contour of anechoic follicles, or for their disappearance anechogenicity by transabdominal ultrasonography and as the proportion of cornified cells was greater than or equal to 90% of epithelial cells from vaginal swabs, which were stained following Diff Quik (Sysmex Co., Kobe, Japan) standard protocols [38].
Oocyte collection
All oocyte donors and surrogates underwent spontaneous estrous, and donors and surrogates were matched based on the synchronization of their estrus. Oocytes were surgically retrieved at 3–4 days post-ovulation. Before surgery, a blood sample was drawn through the cephalic venipuncture, and blood plasma was collected and frozen (− 20 °C) for hormone analyses. Anesthesia was induced with a mixture of xylazine hydrochloride (Rumpun®; Bayer Korea, Ansan, Korea; 1 mg/kg body weight) and ketamine HCl (Ketalar®; Yuhan Corporation; 50 mg/mL, Seoul, Korea; 4 mg/kg body weight) and maintained with isoflurane inhalational. Under aseptic conditions, the reproductive tract was exposed through a midventral incision. Corpora lutea (CL) were counted and oocytes were bilaterally flushed from each oviduct with 10 mL TCM 199 supplemented with HEPES (Invitrogen). Oocytes were collected using a stereomicroscope, transferred into fresh medium, and subjected to nuclear transfer.
Evaluation of retrieved oocytes
The maturation stage of the retrieved oocytes was determined as previously described [39]. The oocytes were stripped of cumulus cells and pre-stained with 5 mg/mL Bisbenzimide (Hoechst 33342) to visualize the presence of nuclei for enucleation process. Oocytes were graded based on their morphology and nuclear stage as immature (cumulus very closely attached to oocytes, nuclear stage is either germinal vesicle (GV), GV breakdown, or metaphase I), mature (M II oocytes with several layers of cumulus cells and homogeneous cytoplasm), aged (unidentified nuclear status with the cytoplasmic membrane shrink, MII oocytes in less than 70% of the cytoplasm and loosely attached cumulus cells,), abnormal (irregular cytoplasmic contour, protrusion of zona pellucida, nuclear immaturity), or ruptured (oocytes with broken zona and cytoplasmic membrane) under an inverted microscope equipped with epiflurescence (TE2000-E; Nikon Corp., Tokyo, Japan).
Preparation of donor cells
Donor cells originated form dogs active in police or military service. Dermal tissue samples from 2 male Belgian Malinois breeds, and 1 male German, measuring approximately 1 × 3 cm were collected under light tranquilization (Zoletil 50® Virbac, Carros, France) at 0.1 mg/kg and local anesthesia (Daehan lidocaine HCl 2%, Dai Han Pharm Co., Ltd., Seoul, Korea). Sections of subcutaneous tissues were cut into small pieces (approximately 1 × mm2) and cultured in DMEM containing 10% FBS at 37 °C in an atmosphere of 5% CO2 and air to obtain fibroblasts. Explants were maintained in the culture until they approached 90% confluence. Cells were then trypsinized and reconstituted at concentrations of approximately 1 × 106 cells per mL, and then cryopreserved in cryovials containing DMEM containing 10% dimethyl sulfoxide.
Nuclear transfer
After evaluating of the maturation status, metaphase II oocytes were enucleated by squeezing out the first polar body and metaphase II plate into a small amount of surrounding cytoplasm using a glass pipette. Donor cells, TP53KO#30 fibroblasts (passage 4), were prepared and treated using a conventional system of primary cell culture as described previously [40]. Using a fine pipette, a trypsinized cells with a smooth cell surface was transferred into the perivitelline space of an enucleated oocyte. The couplets were equilibrated with 0.26 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of CaCl2, and MgSO4 for 4 min. Next, the couplets were transferred to a chamber with two electrodes and covered with mannitol solution. The couplets were fused with two DC pulses of 1.75–1.85 kV/cm for 15 μs using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA, USA). After simultaneous fusion and activation, a group of 5–6 embryos were cultured in 25 μL microdrops of mSOF covered with mineral oil for 1 h at 39 °C in a humidified atmosphere (5% O2, 5% CO2, and 90% N2) until embryo transfer.
Embryo transfer and pregnancy diagnosis
Surrogate dogs with estrus matching that of oocyte donors were anaesthetized as described previously using an oocyte retrieval procedure. The ovary with a greater number of corpus lutuea was approached by ventral laparotomy. The fat layer covering the ovary was gently grasped with forceps and suspended with a suture to exteriorize the fimbriated end of the oviduct. Immediately after fusion and activation, all reconstructed embryos were loaded into a tomcat catheter (3.5 Fr × 5.5″; Sherwood Medical, St. Louis, MO, USA) with at least a medium volume (2-4 μL) and gently transferred into the 2/3 distal position of the oviduct through the infundibulum. Pregnancy was confirmed by transabdominal ultrasound with a real-time ultrasonography at 25–30 days after embryo transfer. Ultrasonography was performed either in the standing or dorsal recumbency position using a portable ultrasound machine with a 3.5 MHz curved transducer (Sonace R7; Samsung Medison, Seoul, Korea). Ultrasonographies were repeated every 7 days on pregnant surrogates until term. The sizes and shapes of the chorionic cavities and presence of an embryonic or fetal heartbeat were examined to identify embryonic or fetal death.
Statistical analysis
All experiments were replicated more than three times. All data were analyzed by one-way ANOVA (analysis of variance) followed by Duncan’s test using SPSS software (SPSS, Inc., Chicago, IL, USA) and are reported as the mean ± standard error of the mean. Differences were considered significant if the P-value was less than 0.05.cancers and even for testing new anti-cancer therapeutics for dogs.