2.1. DNA construction
In previous studies, the transposon system vectors for SB (pCMV(CAT)T7-SB100X) and PB (pCy43 and PB-CA) were purchased from Addgene (http://www.addgene.org, Plasmid #34879 and Plasmid #20960) and/or provided by the Sanger Institute (Hinxton, UK) for the PB and SB systems (26, 27). To newly establish the Tol2 system, transposon and transposase were purchased from Addgene (http://www.addgene.org, Plasmid #97151 and Plasmid #31823). To avoid the backbone sequences effect in application of the transposon systems, all transposon element sequences were amplified by PCR and then PCR products were run for 15 min and extracted by using a Qiagen Gel extraction Kit (Cat No. 28704). Extracted PCR products were cloned with a Qiagen TA cloning kit (Cat No. 231124) (Fig. 1A). All reconstructed vectors were sequenced fully.
2.2. Primary cell culture
For preparation of bovine somatic cells, three types of ear skin were isolated from newborn calves using biopsy punch. After isolation, tissues were collected directly to 50 mL conical tube containing 5 mL of 10% penicillin in PBS to prevent contamination and moved to bench in 3 hours. The tissues were washed 3 times with 10% penicillin-PBS again and minced on 100 mm petri dishes (Falcon, Cat No. 351029) for 5 min and collected in 15 mL conical tubes(SPL, Cat No. 50015) with 5 mL of 10% penicillin-PBS. After centrifugation at 13,000 rpm for 3 min, the supernatant was suctioned off and then the pellet was washed with 5 mL of 10% penicillin-PBS. After 3 times of the centrifugation to washing step, the pellet was suspended with 10 mL of HBSS containing 1% collagenase and incubated at 37°C, 5% CO2 for 17 hrs. After that, samples were centrifuged at 13,000 rpm for 3 min and the pellet was suspended with DMEM (HyClone, Cat No. SH30243.01, USA) containing 20% FBS (Gibco, Cat No. 26140079,USA). The resuspended samples were seeded on 60 mm dish for further cell culture.
2.3. Cell transfection (Electroporation) and FACS sorting
Three types of primary cells were used for electroporation-mediated DNA transfection by using Neon® Transfection system (Invitrogen Cat No. MPK5000). Cell Countess II Automated Cell Counter (Thermo Fisher Scientific) was used to count 3 × 105 cells for transfection. Transfection was replicated 3 times per cell type and transfection conditions were optimization no.16 (1400 V, 20 ms and 2 pulses). Post transfection, transfected cells were seeded directly on incubated 6-well plates containing 3 mL of DMEM. Seventeen hours later, culture media was changed with fresh media to remove dead cells (Fig. 1B).
FACS analysis was conducted to measure the transfection efficiency and integration. To measure the transfection efficiency, sorting was conducted through FACS after 3 days of transfection (Fig. 2A. [a]) and for integration efficiency, sorted cells on day 3 were sub-cultured for 7 days more and re-sorted on day 10 (Fig. 2A. [b]). To conduct FACS sorting, transfected cells were suspended with 500 µL of PBS and the samples were analyzed on BD Bioscience FACS Aria II, installed at the National Center for Inter-university Research Facilities (NCIRF) at Seoul National University.
2.4. In vitro maturation of bovine immature oocytes
Ovaries were collected from a local slaughterhouse within 2-3 h and carried in saline solution 0.9% at 30-35°C during transport to the laboratory. Cumulus–oocyte complexes (COCs) from follicles 2 to 8 mm in diameter were aspirated using an 18-gauge needle, selected, and collected in a 10-cm petri dish. The residue was washed three times with HEPES-buffered tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM NaHCO3 (Sigma–Aldrich Corp., St. Louis, MO, USA), 10% FBS and 1% penicillin–streptomycin (v/v). For in vitro maturation, COCs were cultured in four-well dishes (30–40 oocytes per well; Falcon, Becton-Dickinson Ltd., Plymouth, UK) for 22–24 h in 450 µL TCM-199 supplemented with 0.005 AU/mL FSH (Sigma–Aldrich), 10% FBS, 1 µg/mL 17β-estradiol (Sigma–Aldrich) and 100 µM Cysteamine (Sigma-Aldrich) at 39°C under 5% CO2.
2.5. Sperm preparation, in vitro fertilization (IVF) and in vitro culture of embryos (IVC)
The Percoll gradient method for the separation and purification of motile spermatozoa has been described in detail elsewhere (28). In brief, spermatozoa were refined from thawed semen straws by density-gradient centrifugation on a Percoll discontinuous gradient (45–90%) at 1680 rpm for 15 min. For the Percoll density gradient, the 45% Percoll solution was prepared with 1 mL of 90% Percoll (Nutricell, Campinas, SP, Brazil) and 1 mL of capacitation-TALP (Nutricell) (29). and then 1 mL of 45% Percoll solution was added onto 1 mL of 90% Percoll solution in a 15 mL conical tube. The thawed semen was layered onto the top of the Percoll gradient solution and the tube was centrifuged. The pellet was washed twice with 3 mL of TALP by pipetting briefly and centrifugation for 5 min at 1680 rpm. The active, motile spermatozoa from the pellet were added to droplets containing matured oocytes. Oocytes were inseminated on (day 0) with 1–2 × 106 spermatozoa/mL for 17 h in IVF-TALP medium (Nutricell) under NidOil (Nidacon). The fertilized zygotes were denuded and cultured in two-step defined culture medium (4 days in D1 medium before transfer to D2 medium) at 39°C in an atmosphere of 5% O2, 5% CO2 and 90% N2 (30). Cleavage rates were recorded on Day 4 and embryonic development was monitored according to the stages of the International Embryo Transfer Society (IETS).
2.6. Microinjection
Microinjection experiments were conducted through three different transposon systems to see whether the stable transposon systems in somatic cells could be used at the bovine embryo stage. After 17 h in IVF, denuded zygotes were used for microinjection (Femtojet®, Eppendorf, Germany). To find out the optimal microinjection conditions, 2 different DNA concentrations were assessed (High: 50 ng/µL, Low: 25 ng/µL). Six days later, GFP-expressing blastocysts were observed with a fluorescent microscope and stained with Hoechst 33342 (Sigma Aldrich) to count total cell number in blastocysts (Fig. 1C, Fig. 3, and Table 1).
2.7. Statistical Analysis
Data obtained from each of three replicated experiment results were statistically analyzed using One-way ANOVA followed by Tukey’s multiple comparisons test was performed using GraphPad Prism version 7.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com, The results were considered statistically significant when the p value was equal to or lower than 0.05.