Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Animal care and chemicals

All of the animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Meiji University (IAUCU-12-0008). Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.

Construction of the Plum expression vector

The Plum expression vector used in the present study consisted of a chicken beta-actin promoter with a cytomegalovirus enhancer (CAG promoter), Plum cDNA, rabbit beta-globin 3’-flanking sequence including a polyadenylation signal, and the puromycin N-acetyltransferase gene driven by the phosphoglycerate kinase (PGK) promoter (Fig. 1A). The Plum expression vector was constructed based on the pCX-GFP vector [38]. Briefly, Plum cDNA was amplified by polymerase chain reaction (PCR) from a plasmid containing the Plum coding sequence (pmPlum Vector, Takara Bio, Shiga, Japan). The amplified product was inserted into the EcoRI restriction sites of the pCX-GFP vector to replace the GFP fragment with Plum using a cloning kit (In-Fusion HD Cloning Kit, Takara Bio) to produce pCX-Plum. For antibiotic selection, a 1.1-kb fragment including the puromycin N-acetyltransferase gene under the control of the PGK promoter (puroR) was inserted into the HindIII site of the constructed pCX-Plum vector. Finally, the constructed Plum expression vector, designated pCX-Plum-puroR (Fig. 1A), was verified by sequencing using a 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA). The transgene fragment was excised from the plasmid vector by enzymatic digestion using SalI (Takara Bio) and BamHI (Takara Bio), separated by gel electrophoresis, and purified using a gel extraction kit (QIAquick Gel Extraction Kit, QIAGEN, Hilden, Germany).

Preparation of nuclear donor cells

A primary culture of porcine female fetal fibroblasts [39] was prepared for nuclear donor cells. The porcine fetal fibroblasts (PFFs) were cultured in minimum essential medium (MEM Alpha, Life Technologies) supplemented with 15% fetal bovine serum (FBS, Nichirei Bioscience, Tokyo, Japan) and antibiotic-antimycotic solution (Life Technologies) with type I collagen-coated dishes (AGC Techno Glass, Shizuoka, Japan) in a humidified atmosphere containing 5% CO₂ at 37 C.

For transfection, PFFs were cultured to 70–90% confluence, washed twice with Dulbecco’s phosphate-buffered saline (DPBS) and collected after treatment with 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA, Life Technologies). The collected cells (6.0 × 10⁵) were then resuspended in 60 µl of resuspension buffer supplied as part of a Neon Transfection System kit (Life Technologies), and 1.5 µg of pCX-Plum-puroR was added. The cells were then electroporated under the following conditions: pulse voltage, 1,100 V; pulse width, 30 ms ; and pulse number, 1. Forty-eight hours after electroporation, the cells were transferred to medium containing 2.5 µg/ml puromycin. At 12 days in culture, Plum-positive cells (1.0 × 10⁵ cells) were collected from puromycin-resistant cells using a BD FACSAria III cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) equipped with a 561-nm (Yellow-Green) laser and were seeded onto a collagen-coated dish (AGC Techno Glass). Plum-positive cells (Plum-PFFs) were grown to confluence within 2–3 days and were then cryopreserved for later use as nuclear donor cells to generate Tg fetuses expressing Plum.

For chromosome counting, metaphase chromosome spreads from pig fibroblast cells were prepared according to standard procedures [40]. Cells were treated with 20 ng/ml colcemid (demecolcine) for 14 h and harvested. After treatment with 0.075 M KCl for 20 min at room temperature (RT), cells were fixed by exposure to MeOH:acetic acid (3:1) three times, and fixed cells were spread on slides. Chromosome images were captured using a Leica DC350FX cooled CCD camera (Leica, Wetzlar, Germany) mounted on a Leica DMRA2 microscope and analyzed using the Leica CW4000 FISH software.

SCNT

SCNT was performed as described previously [23] with slight modifications. Briefly, in vitro matured oocytes containing the first polar body were enucleated by gentle aspiration of the polar body and the adjacent cytoplasm using a beveled pipette in Tyrode lactose medium containing 10 mM HEPES and 0.3% (w/v) polyvinylpyrrolidone (HEPES-TL-PVP) in the presence of 0.1 µg/ml demecolcine, 5 µg/ml cytochalasin B (CB) and 10% FBS.

Nuclear donors were used following cell cycle synchronization by serum starvation for 2 days. A single donor cell was inserted into the perivitelline space of an enucleated oocyte. The donor cell-oocyte complexes were placed in a solution of 280 mM mannitol (pH 7.2; Nacalai Tesque, Kyoto, Japan) containing 0.15 mM MgSO₄, 0.01% (w/v) polyvinyl alcohol (PVA) and 0.5 mM HEPES and then held between two electrode needles. Membrane fusion was induced with a somatic hybridizer (LF201, Nepa Gene, Chiba, Japan) by applying a single direct-current (DC) pulse (267 V/mm, 20 µsec) and a pre- and post-pulse alternating current (AC) field of 2 V at 1 MHz for 5 sec. The reconstructed embryos were cultured in porcine zygote medium-5 (PZM-5; Research Institute for the Functional Peptides, Yamagata, Japan) supplemented with 4 mg/ml bovine serum albumin (BSA) for 1–1.5 h, followed by electrical activation. For induction of electrical activation, the reconstructed embryos were aligned between two wire electrodes (1.0 mm apart) of a fusion chamber slide filled with activation solution consisting of 280 mM mannitol, 0.05 mM CaCl₂, 0.1 mM MgSO₄ and 0.01% (w/v) PVA. A single DC pulse of 150 V/mm was applied for 100 µsec using an electrical pulsing machine (Multiporator, Eppendorf, Hamburg, Germany). After activation, the reconstructed embryos were cultured in PZM-5 for 3 h in the presence of 5 µg/ml CB and 500 nM Scriptaid, followed by culture with 500 nM Scriptaid for another 12–15 h. After these treatments, the cloned embryos were cultured in PZM-5 for 7 days to assess their in vitro development.

Embryo culture was performed under a humidified atmosphere of 5% CO₂, 5% O₂ and 90% N₂ at 38.5 C. Beyond the morula stage, the embryos were cultured in PZM-5 supplemented with 10% FBS.

Transfer of cloned embryos into recipient pigs

Crossbred prepubertal gilts (Large White/Landrace × Duroc) weighing 100–105 kg were used as recipients of the cloned embryos. The gilts were given a single intramuscular injection of 1,000 IU of equine chorionic gonadotropin (eCG, ASKA Pharmaceutical, Tokyo, Japan) to induce estrus. Ovulation was induced by an intramuscular injection of 1,500 IU of human chorionic gonadotropin (hCG, Kyoritsu Pharmaceutical, Tokyo, Japan) given 66 h after the injection of eCG. The cloned embryos were cultured for 5 or 6 days and were surgically transferred into the uterine horns of the recipients approximately 146 h after hCG injection.

Generation of Plum-expressing offspring and analysis of the embryos, fetuses and offspring

We analyzed the expression of the Plum in Tg embryos at the one-cell (day 1), two-cell (day 2), four-cell (day 2), eight-cell (day 3), morula (day 4) and blastocyst (days 5 and 7) stages using confocal microscopy (FV-1000, Olympus, Tokyo, Japan). Some of the day-7 blastocysts were mounted on glass slides (Matsunami Glass Ind., Osaka, Japan) in HEPES-TL-PVP containing 20% ethylene glycol (Nacalai Tesque) and 5 mg/ml Hoechst 33342; these embryos were examined by fluorescence microscopy (TE-300 microscope, Nikon, Tokyo, Japan) to count cell numbers.

Recipient pigs to which SCNT embryos had been transferred were euthanized at day 36–37 of gestation, and the fetuses were collected. These fetuses were used to confirm the expression of Plum and to determine transgene copy numbers; additionally, the fetuses were used to generate rejuvenated porcine fetal fibroblasts (Neo Plum-PFFs) for the next round of SCNT to produce offspring. We used SCNT to generate Plum-expressing offspring using Neo Plum-PFFs as nuclear donor cells. On day 2 after birth, we also screened for Plum expression in the tissues and organs of piglets. Fluorescence was analyzed using a fluorescence microscope (MVX10, Olympus; excitation, 532.5–587.5 nm; emission, 607.5–682.5 nm).

Flow cytometric analysis of blood cells from Tg pigs expressing Plum was performed using a BD FACSAria III cell sorter. Freshly isolated blood cells were lysed with BD Pharm Lyse (Becton, Dickinson and Company) reagent to remove erythrocytes. Each cell population was selected by gating strategies based on forward and side scatter properties.

Female Tg cloned offspring were euthanized at 10 months of age. The ovaries were removed, and cumulus-oocyte complexes were collected as described previously [23]. The expression of Plum in the cumulus-oocyte complexes was confirmed using a confocal microscope (FV-1000, Olympus).

Estimation of transgene copy number by Southern blot analysis

Genomic DNA was extracted from skin samples of Tg cloned fetuses using a DNA purification kit (DNeasy Blood & Tissue Kit, QIAGEN). The purified genomic DNA (5 µg) was digested with PstI (Takara Bio), separated by gel electrophoresis, and transferred onto a nylon membrane (Hybond N⁺, GE Healthcare Bio-Sciences, Uppsala, UK). The membranes were blocked for 30 min at room temperature with blocking reagent (Blocking One, Nacalai Tesque). After blocking, the membranes were incubated in hybridization solution (DIG Easy Hyb, Roche Diagnostics, Basel, Switzerland) and hybridized with a digoxigenin (DIG)-labeled Plum probe prepared by PCR using a DNA-labeling reagent (DIG DNA Labeling Mix, Roche Diagnostics). The blot was developed using chemiluminescent reagent (DIG Luminescent Detection Kit, Roche Diagnostics), and the signal was detected and imaged with an ImageQuant LAS-4000 system (GE Healthcare Bio-Sciences). The number of transgene copies integrated into the porcine genome was determined by comparison of the hybridization signal with that of the copy-number control, which was diluted to make a standard series (1–10 copies per diploid genome).

Multicolor fluorescence imaging using Plum

To determine whether Plum-expressing PFFs could be distinguished from PFFs expressing different fluorescent proteins (e.g., EGFP and huKO), we carried out both in vitro fluorescence observations and flow cytometry analyses. PFFs that express EGFP [36] and huKO [22] were generated previously; we cocultured these PFFs with fibroblasts derived from Plum-expressing fetuses. The three cell types were inoculated onto a 35-mm dish (7.0 × 10⁴ cells for each cell type) and observed by confocal microscopy (FV-1000, Olympus) at 24 h after the start of culture. To confirm that the three cell types could be fractionated using their different fluorescence colors, a cell mixture (1.0 × 10⁶ cells of each cell type) was sorted using a BD FACSAria III cell sorter. For each cell type, 2.0 × 10⁵ cells were sorted and inoculated onto a 35-mm dish; 24 h later, cells were screened using a confocal microscope (FV-1000, Olympus). The excitation wavelengths and emission wavelengths of the three fluorescent proteins were as follows: EGFP, 488 nm/495–515 nm; huKO, 559 nm/561–610 nm; and Plum, 559 nm/ 650–750 nm.

Statistical analysis

Experimental results were expressed as the mean ± standard error of the mean (SEM). The data were analyzed using the SPSS 16.0 software (SPSS, Chicago, IL, USA). For proportional data, differences between groups were analyzed using the χ² test. For blastocyst cell number data, differences between groups were determined by Student’s t-test. The level of significance was set at P < 0.05.

Generation of Plum-expressing SCNT embryos

Plum-PFFs obtained by transferring pCX-Plum-puroR showed no abnormalities in shape or chromosome number (Fig. 1). The cleavage and blastocyst formation rates in SCNT embryos reconstructed using Plum-PFFs (Plum embryos) were 81.0% (34/42) and 78.6% (33/42), respectively. In comparison, the rates for control SCNT embryos reconstructed using PFFs without the Plum gene were 92.5% (37/40) and 75.0% (30/40), respectively (Table 1). The average number of cells in Plum blastocysts was 88.3 ± 6.0 compared with 99.9 ± 8.8 in control blastocysts. Our results indicated there was no significant difference in in vitro development ability between the two groups of embryos (P < 0.05).

Analysis of fluorescence in Plum embryos confirmed weak expression of Plum at the eight-cell stage. Higher expression was observed as development progressed (Fig. 2).

Production and analysis of Plum-expressing cloned fetuses

One hundred three SCNT embryos reconstructed using Plum-PFFs as nuclear donor cells were transferred to a single recipient pig; three fetuses were obtained at 36–37 days of gestation (3/103, 2.9%) (Table 2). Plum fluorescence was observed systemically in all three fetuses and was visible in fibroblasts (Neo Plum-PFFs) derived from the fetuses. In contrast, wild-type fetuses showed only autofluorescence (Fig. 3). Southern blot analysis revealed that Plum fetuses had 1–5 copies of the transgene (Fig. 3E).

Characterization of Plum-expressing cloned offspring

To generate offspring expressing Plum, we used Neo Plum-PFFs from one of the three cloned fetuses (Fig. 3D and D’) as nuclear donor cells. One hundred seventeen SCNT embryos were transferred to a single recipient pig; four cloned offspring and one stillborn cloned offspring were produced (5/117, 4.3%) (Table 2). At birth, the piglets weighed 911.0 ± 115.8 g and had a body length of 27.3 ± 1.3 cm. The offspring developed normally to adulthood. Expression of Plum protein was confirmed in lymphocytes, monocytes and granulocytes and in 15 different tissues and organs (Figs. 4 and 5). Plum expression was identified in cumulus-oocyte complexes from the ovaries collected after euthanasia at 10 months of age (Supplementary Fig. 1: online only).

Function of Neo Plum-PFFs in cells expressing other fluorescent proteins

The three types of PFFs expressing EGFP [37], huKO [23] and Plum were clearly distinguished in culture by confocal microscopy based on excitation wavelengths and emission wavelengths. In addition, using a flow cytometer, we found that a mixture of the three cell types was distributed at wavelengths for EGFP, huKO and Plum (Fig. 6B). Subsequently, the three cell types were precisely sorted at the appropriate wavelength ranges corresponding to the emission wavelength of the expressed fluorescent protein (Fig. 6C).