Development of a Noninvasive Monitoring System for Evaluation of ...

Journal of Reproduction and Development, Vol. 55, No. 6, 2009, 09-089A

—Full Paper—

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos Kazuchika MIYOSHI1), Hironori MORI1), Yamato MIZOBE1), Eri AKASAKA2), Akio OZAWA2), Mitsutoshi YOSHIDA1) and Masahiro SATO2) 1) Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065 and 2)Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-0065, Japan

Abstract. The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4–6 days of culture) and 50.0% of blastocysts (at 5–6 days of culture), whereas none of the 1cell to 16-cell embryos at 1–5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4–6 days after SCNT and that this noninvasive monitoring system using Oct3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei. Key words: Enhanced green fluorescent protein, In vitro development, Nuclear transfer, Oct-3/4 promoter, Reprogramming (J. Reprod. Dev. 55: 661–669, 2009) uccessful production of cloned animals using somatic cell nuclear transfer (SCNT) has been reported in several mammalian species [1–12]. However, the low cloning efficiency associated with development of SCNT embryos to offspring remains the major obstacle to widespread use of this technology in a number of animal science and biomedical applications. A method for evaluating the developmental ability of SCNT embryos before being transferred into recipient females should be explored to optimize the procedures of SCNT and overcome the low cloning efficiency. Evaluation of the in vitro developmental ability of an SCNT embryo to the blastocyst stage would be considered one of the methods for predicting its in vivo development, but it has been found that this does not often assure successful development to term of SCNT embryos [13]. After SCNT, the gene expression pattern in somatic cells is reprogrammed to mimic that in preimplantation embryos [14]. However, there is a difference in gene expression pattern between SCNT embryos and in vitro-fertilized embryos, suggesting that the low efficiency of SCNT is associated with incomplete reprogramming in the donor nuclei transferred into recipient oocytes [15]. Therefore, evaluation of the reprogramming level in the donor nuclei appears to be most essential for predicting the in vivo developmental ability of SCNT embryos. Oct-3/4 is a transcription factor of the Pit-Oct-Unc family [16, Accepted for publication: August 28, 2009 Published online in J-STAGE: October 1, 2009 Correspondence: K Miyoshi (e-mail: [email protected])

17], and its expression starts from the 2-cell stage in mouse embryos [18]. Although Oct-3/4 is expressed initially in all blastomeres during embryonic development, its expression becomes restricted to the inner cell mass (ICM) at the blastocyst stage and concomitantly is down-regulated in the trophectoderm (TE) [19, 20]. Subsequently, the expression of Oct-3/4 is strictly confined to primordial germ cells (PGCs), the precursors of oocytes and spermatozoa [21]. Oct-3/4-deficient mouse embryos develop into blastocysts without a pluripotent ICM and fail to implant, suggesting a key role for Oct-3/4 in maintenance of pluripotent cell populations [22]. Oct-3/4 is also required for pluripotency and self-renewal of embryonic stem (ES) cells derived from the ICM [23] and is abundantly expressed in ES cells as well as embryonal carcinoma (EC) cells that exhibit more restricted ability to differentiate than ES cells [24, 25]. The expression of Oct-3/4 in preimplantation embryos has also been reported in cattle and pigs [26–28]. In addition, Oct-3/4 expression patterns in the tissues of adult pigs are the same as those in adult mice; Oct-3/4 is expressed in the PGCs, ovary and testis, but not in the heart, liver, lung, kidney, spleen and fibroblasts [29]. These results indicate that Oct-3/4 expression is strictly regulated during at least preimplantation embryonic development, suggesting that reactivation of Oct-3/4 gene in the donor nuclei would be a suitable indicator for evaluating the reprogramming level of SCNT embryos. In mice, transgenic animals expressing green fluorescent protein (GFP) under the control of Oct-3/4 promoter have been produced that allow the real-time observation of PGCs in living embryos

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[30]. In addition, the reprogramming level of embryos reconstituted with their somatic cells can be evaluated by GFP expression [31]. The objective of the present study was to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig SCNT embryos during in vitro development using a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced GFP (EGFP) gene and a neomycin resistance gene (neo).

Materials and Methods

coated dishes (Iwaki Glass, Tokyo, Japan) at a density of 1 × 106 cells/well 1 day before transfection and grown in DMEM supplemented with 10% FBS at 37 C in an atmosphere of 5% CO2 in air. For transfection, 2 μg of circular pOEIN or pmaxGFP DNA was mixed with 4 μl of FuGENE6 (Roche, Basel, Switzerland) in Dulbecco’s modified phosphate-buffered saline without Ca2+ and Mg2+, pH 7.4 [PBS(–)], and a total of 100 μl of solution was prepared according to the manufacturer’s protocol. The DNA/ liposome complexes were added to the cell culture and incubated for 1 day at 37 C. After transfection, cells were observed for fluorescence under ultraviolet (UV) light.

Plasmids For construction of pOEIN plasmid (Fig. 1A), the mouse Oct-3/ 4 promoter region spanning 5.4 kb was isolated by digestion of pGOF-18 [21] with Sal I and Spe I and placed upstream of EGFP cDNA + poly(A) sites of SV40 gene in pEGFP-N1 (Clontech, Palo Alto, CA, USA) to create pOE. On the other hand, a 1.2-kb HS4 insulator from rabbit β-globin gene [32] was ligated to upstream of 1.5-kb neo expression unit in which expression of neo was driven by mouse phosphoglycerate kinase promoter to create pIN. Construction of pOEIN was accomplished by ligation between OE [comprising Oct-3/4 promoter + EGFP cDNA + poly(A) sites] and IN (comprising HS4 insulator + neo expression unit) in a cloning vector, pBluescript SK(–) (Stratagene, La Jola, CA, USA). The fidelity of pOEIN was confirmed by restriction enzyme analysis and sequencing. pmaxGFP (Amaxa GmbH, Cologne, Germany), which carries cytomegalovirus promoter and the copepod Pontellina sp.-derived GFP cDNA, was used as a positive control to examine the transient expression system. These plasmids were amplified in DH5αE. coli and purified using a Qiagen plasmid DNA isolation kit (Qiagen GmbH, Hilden, Germany). Circular plasmid DNA was used for transient expression assay, and pOEIN was linearized by digestion with Not I and used for acquisition of stable transfectants.

Cells Miniature pig fetal fibroblasts (MPFFs) were obtained from a male Clawn miniature pig fetus on Day 30 and primarily cultured in a 100-mm plastic dish (Nunc, Roskilde, Denmark) containing a mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% (v:v) fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 50 units/ml penicillin and 50 μg/ ml streptomycin (MPFF culture medium) at 37 C in a humidified atmosphere of 5% CO2 in air. The cells were passaged 3–4 times and then frozen using Cell Banker (Wako). At experiment, the frozen cells were thawed and passaged 3–7 generations prior to transfection. Cells of the F9 EC line [33], a nullipotent mouse cell line that is incapable of differentiation under normal conditions, and NIH3T3, a cell line derived from mouse fetal fibroblasts, were cultured in DMEM (Invitrogen) supplemented with 10% FBS at 37 C in an atmosphere of 5% CO2 in air.

Transient expression assay F9 and NIH3T3 cells were first seeded onto 6-well gelatin-

Establishment of stable MPFF clones carrying pOEIN plasmid To obtain stable pOEIN transfectants, MPFFs were transfected with linearized pOEIN DNA using a nucleofector-mediated transfection system (Amaxa) and the method of Nakayama et al. [34], with some modifications. Transfection was performed in a total volume of 100 μl containing 1 × 106 MPFFs, 10 μg of DNA and Amaxa nucleofector solution specifically formulated for transfection of primarily cultured cells using an Amaxa cuvette with a 2.5 mm gap. Cells were then nucleofected using a preprogrammed setting (B16) on the Amaxa nucleofector apparatus. After electroporation, cells were seeded onto 3 gelatin-coated 100-mm dishes (IWAKI) containing 6 ml of MPFF culture medium. At 2–4 days of culture, the medium was replaced with MPFF culture medium containing 400 μg/ml G418 (BioLinks, Tokyo, Japan), and the cells were cultured for an additional 7–10 days. Colonies exhibiting G418 resistance were picked up using a small paper disc that had been dipped in 0.125% (w:v) trypsin/0.01% (w:v) EDTA and propagated in a 48-well dish (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 0.8 ml of MPFF culture medium supplemented with G418 for an additional 10–20 days. Stable transformants were further propagated in a 24-well dish (IWAKI) and then frozen. A portion of each clone was subjected to PCR analysis for confirmation of the presence of pOEIN transgene in its genome, as described below. One of these clones, pOEINMPFF-#4, was used as the donor for SCNT between passages 10 and 13 of culture. The cells were allowed to grow to confluency and continued to culture for an additional 5–6 days without changing the medium. A single cell suspension was prepared by standard trypsinization immediately prior to SCNT, and the cells were inspected for expression of EGFP under UV light.

In vitro maturation of oocytes Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in saline at 32–36 C. The follicular contents were recovered by aspiration from follicles (2–5 mm in diameter) using an 18-gauge needle (Terumo, Tokyo, Japan) and a 5-ml disposable syringe (Nipro, Osaka, Japan). The cumulus-oocyte complexes (COCs) were gathered from the follicular contents and washed twice with HEPES (Nacalai Tesque, Kyoto, Japan)-buffered Tyrode-lactate-pyruvate-polyvinyl alcohol (PVA; Sigma-Aldrich Chemical, St. Louis, MO, USA) and the maturation medium, respectively. Only COCs possessing a compact cumulus mass and evenly granulated ooplasm were selected. COCs in groups of 30–50 were transferred to a droplet of the maturation

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medium (200 μl) under paraffin oil (Nacalai Tesque) in a 35-mm dish (Becton, Dickinson and Company) and cultured at 38.5 C in an atmosphere of 5% CO2 in air. The maturation medium consisted of 90% (v:v) TCM-199 with Earle’s salts (Gibco BRL, Grand Island, NY, USA) supplemented with 0.91 mM sodium pyruvate (Sigma), 3.05 mM D-glucose (Wako), 0.57 mM cysteine hydrochloride hydrate (Sigma), 10 ng/ml epidermal growth factor (Sigma), 10 IU/ ml eCG (Aska Pharmaceutical, Tokyo, Japan), 10 IU/ml hCG (Aska), 100 μg/ml amikacin sulfate (Meiji Seika, Tokyo, Japan), 0.1% (w:v) PVA and 10% (v:v) pig follicular fluid. After 40–42 h of culture, cumulus cells were removed by pipetting with 0.1% (w:v) hyaluronidase (Sigma). Oocytes with a polar body were selected for the experiments.

Production of SCNT embryos HEPES-buffered TCM-199 with the osmolarity adjusted to 300 mOsm by adding sucrose supplemented with 10% FBS was used as the basic medium (BM) for SCNT. In vitro-matured and denuded oocytes were cultured in 100 μl of BM supplemented with 0.2 μg/ ml demecolcine (Sigma) and 20 mM sucrose for 0.5–1 h [35]. Oocytes with a protruding membrane were transferred into BM supplemented with 0.2 μg/ml demecolcine and 5 μg/ml cytochalasin B. The protrusion was removed by aspiration with a 15-μm inner diameter glass pipette. A single pOEIN-MPFF-#4 cell was inserted into the perivitelline space of each enucleated oocyte using the same glass pipette. Cell-oocyte complexes were transferred to BM and kept in 5% CO2 in air at 38.5 C until fusion. The chamber for fusion was a 60-mm dish (Becton, Dickinson and Company) filled with 7 ml of fusion medium composed of 250.3 mM sorbitol, 0.5 mM Mg(CH3COO)2, 0.3 mM HEPES and 0.2% (w:v) BSA. Two stainless-steel wires (100 μm in diameter) were used as electrodes and were attached to micromanipulators. A single cell-oocyte complex was sandwiched between the electrodes and oriented with the contact surface between the cytoplast and donor cell perpendicular to the electrodes. Membrane fusion was induced by applying a single direct-current pulse of 25 V for a duration of 20 μsec with a prepulse alternating-current field of 5 V, 1 MHz, for 2 sec using an LF 101 Fusion Machine (Nepa Gene, Chiba, Japan). Following the fusion pulse, the complexes were cultured for 2 h in 100 μl of modified porcine zygote medium-3 (mPZM-3) [36] until activation. Fusion was determined by microscopic examination 1 h after applying the pulse.

Activation and culture of embryos and oocytes Fused embryos and in vitro-matured oocytes were activated by ultrasound stimulation [36–38]. Briefly, the embryos and oocytes in groups of 30–60 were washed twice in activation medium composed of 250.3 mM sorbitol, 0.5 mM Ca(CH 3COO)2 , 0.5 mM Mg(CH3COO)2 and 0.1% BSA [39] and then transferred to a well of a 4-well dish (Nunc) containing 800 μl of the same medium. The ultrasound probe (8 mm in diameter) of a KTAC-3000 Sonopore (Nepa Gene) was inserted directly into the activation medium, and the embryos were exposed to 2872-kHz ultrasound at an intensity of 65 V for 30 sec with a 10-Hz burst rate and 10% duty cycle. The oocytes were exposed to the same ultrasound except that the intensity and duty cycle were changed to 45 V and 30%, respec-

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tively. A miniature stirrer was placed within the well and spun at 300 rpm during ultrasound exposure. After exposure, the embryos and oocytes were cultured in 50 μl of mPZM-3 under 5% CO2, 5% O2 and 90% N2 at 38.5 C. During the first 2 h of culture, 2.2 μg/ml cytochalasin B was added to the medium to prevent extrusion of a polar body-like structure or second polar body. The embryos were cultured for 1, 2, 3, 4, 5, 6 or 7 days and then assessed for developmental stages and expression of EGFP under UV light. Some embryos were used for immnohistochemical staining and identification of the transgene, as described below. The oocytes were cultured for 5 days, and the resultant morulae and blastocysts were also observed under UV light as controls for the expression of EGFP. In addition, some oocytes were cultured for 7 days, and the resultant blastocysts were used as controls for identification of the transgene in the embryos.

Immunohistochemistry SCNT embryos were first fixed with PBS(–) containing 4% (v:v) paraformaldehyde for 5 min at room temperature, permeabilized by immersing them in PBS(–) containing 0.1% (v:v) Triton X-100 and 10% FBS for 10 min at room temperature, washed twice with PBS(–) containing 1% FBS [PBS(–)/FBS] and then immersed in a drop (ca. 20 μl) of PBS(–)/FBS containing 5 μg/ml goat antiOct-3/4 antibodies (C-20, #SC-8629; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a well of a Terasaki microtest plate (Nunc). The drop was covered with paraffin oil to avoid evaporation of the solution. Reaction was performed overnight at 4 C. The embryos were then washed twice with PBS(–)/FBS and subsequently immersed in a drop (ca. 20 μl) of PBS(–)/FBS containing 4 μg/ml Alexa Fluor 594-labelled rabbit anti-goat IgG antibodies (#A11080; Invitrogen) in a well of a Terasaki microtest plate for 2 h at 4 C.

Observation of fluorescence Fluorescence (EGFP-derived green and/or Alexa Fluor 594derived red fluorescence) in SCNT embryos and parthenogenetically developed embryos was observed using a fluorescent microscope (Nikon, Tokyo, Japan) with an attached digital camera (Nikon) and photographed. The data incorporated into a Macintosh computer were processed using the Adobe Photoshop 5.0 software (Adobe System, Seattle, WA, USA). For measurement of the intensity of fluorescence in embryos, at least 4 points exhibiting maximal levels of fluorescence were selected. As a negative control, fluorescence levels at an arbitrarily selected area outside the embryo were concomitantly measured. Thus, the degree of fluorescence (herein referred to as “intensity of fluorescence=IF”) in the embryos was expressed as the intensity of fluorescence in the embryo/the intensity of fluorescence in the area outside the embryo. IF between 1.00 and 1.10 was defined as negative for fluorescence.

Isolation of genomic DNA from cultured MPFFs Genomic DNA of transfected cells was isolated as previously described [40] with several modifications [41]. Cells (ca. 5 × 105) were digested by incubating them with 150 μg/ml proteinase K (Merck, Darmstadt, Germany) and 0.5 mg/ml Pronase E (Kaken

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Kagaku, Tokyo, Japan) in 500 μl of lysis buffer (100 mM NaCl, 0.4% SDS, 10 mM Tris buffer and 10 mM EDTA, pH 8.0) at 37 C for 1 day. An equal volume of saturated phenol was added, and the mixture was extracted once. The aqueous phase was then precipitated with isopropanol. The DNA threads generated were picked up using a Gilson tip, washed with 70% (v:v) ethanol and air-dried. Distilled water was added to each sample, and the DNA was quantitated spectrophotometrically.

Whole genome amplification (WGA) of blastocyst DNA Each blastocyst obtained after culture for 7 days was placed in a 10 μl of mPZM-3 in a 200 μl-microfuge tube and immediately deep-frozen. For lysis of blastocysts, 10 μl of lysis buffer containing the Pronase E and proteinase K mentioned above was added to each microfuge tube containing a single blastocyst and incubated at 37 C overnight. WGA was performed using a GenomiPhi kit (Amersham-Pharmacia) according to the manufacturer’s protocol. Briefly, 2 μl of lysed solution was suspended in 8 μl of a sample buffer, denatured at 95 C for 3 min and cooled to room temperature. A reaction buffer (9 μl) and enzyme mix (1 μl) were added to the denatured sample, and the reaction mixture was incubated at 30 C for 18 h. The sample buffer, reaction buffer and enzyme mix were all components of the kit. The reaction was stopped by heat inactivation at 65 C for 10 min. A portion (2 μl) of this product was subjected to PCR and subsequent nested PCR analyses, as described below.

PCR analysis PCR amplification reactions were performed in a total volume of 10 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.25 mM each of dATP, dCTP, dGTP and dTTP, 1 mM of sense and reverse primers, 2 μl of genomic DNA and 0.5 units Taq polymerase (Takara Shuzo, Tokyo, Japan). The primer set [EGFP5S (5’-gCg ATg CCA CCT ACg gCA AgC-3’) and EGFP-5RV (5’-gAg CTg CAC gCT gCC gTC CTC-3’)] [42] corresponds to the middle portion of EGFP cDNA. For amplification of genomic DNA from cultured MPFFs, 2 μl (ca. 0.5 μg) of DNA was subjected to PCR. Two μl of pOEIN plasmid (5 ng) was used as a positive control when genomic DNA from cultured MPFFs was examined. Similarly, 2 μl of genomic DNA (ca. 0.5 μg) from nontransfected MPFFs was used as a negative control. For amplification of blastocyst-derived DNA, 2 μl of solution used for WGA using a GenomiPhi kit was directly subjected to PCR. Two μl of genomic DNA (ca. 10 pg) from pOEIN-MPFF-#4 cells (positive control) or non-transfected MPFFs (negative control) was subjected to WGA and subsequently to PCR. Thirty two-cycles of PCR reactions were performed in a Astec Program Temp Control System PC-800 with cycle times of 1 min at 94 C, 1 min at 56 C and 2 min at 72 C. This PCR yielded 431-bp fragments from the EGFP cDNA. For nested PCR, the 1st PCR products (2 μl) were mixed with the PCR reaction solution mentioned above, except for use of a nested primer set [EGFP-7S (5’-CAA gCT gAC CCT gAA gTT CAT-3’) and EGFP-7RV (5’-gTC CTC gAT gTT gTg gCg gAT-3’)] in a total volume of 10 μl. The reaction conditions were the same as for the 1st PCR. This PCR yielded 400-bp fragments from the EGFP

cDNA. Products of the reaction were analyzed by electrophoresis on a 2% (w:v) agarose gel. The gels were stained with EtBr, and the amplified DNA bands were visualized by UV illumination.

Genomic Southern blot analysis Genomic Southern blot analysis for cultured transfectants was performed as described previously with slight modifications [23]. Genomic DNA (5 μg) isolated from transfectants or non-transfected MPFFs was digested with Eco RI and Bsr GI, which cleave pOEIN DNA twice and release an approximately 2-kb fragment containing a portion of Oct-3/4 promoter and the entire cDNA for EGFP (Fig. 1A). A 9.5-pg portion of pOEIN DNA was calculated to be equivalent to one copy of DNA per diploid cell based on a genome size of 6 × 109 bp per diploid cell. Based on this calculation, purified pOEIN DNA was added to non-transfected MPEF DNA to obtain various copy levels of pOEIN DNA and processed concomitantly with the experimental samples.

Results Transient expression assay To examine whether mouse Oct-3/4 promoter in pOEIN plasmid would exhibit tissue specificity, a transient expression assay was first performed using mouse F9 and NIH3T3 cells as tester cells. F9 cells are nullipotent undifferentiated EC cells expressing Oct-3/ 4, while NIH3T3 cells are differentiated fibroblastic cells that do not express Oct-3/4. Transfection with circular pmaxGFP resulted in efficient expression of EGFP in both cell types, but only F9 cells expressed EGFP upon transfection with pOEIN (Fig. 1B). This result clearly shows that pOEIN is useful for reporting the presence of Oct-3/4-expressing cells.

Establishment of stable transfectants carrying pOEIN After 7–10 days of selection with G418, a total of 12 G418-resistant MPFF clones were isolated. PCR analysis of genomic DNA isolated from these clones revealed that all the samples tested had pOEIN transgenes in their genome (Lanes 1–12 in Fig. 2A). As expected, no EGFP-derived fluorescence was seen in any clones after inspection under UV illumination (data not shown). Of these stable transfectants, we chose pOEIN-MPFF-#4 (corresponding to Lane 4 in Fig. 2A), since it proliferated relatively faster than the other clones. Genomic Southern blotting revealed that this clone had about 5 copies of pOEIN in its genome (Fig. 2B).

EGFP expression in SCNT embryos cultured in vitro The pOEIN-MPFF-#4 cells did not express EGFP under UV light immediately prior to SCNT (Fig. 3A and E). At 1 day of culture after SCNT of donor nuclei from pOEIN-MPFF-#4 cells, 57.1% of embryos developed to the 2-cell to 4-cell stages (Table 1). Most (57.1%) of the embryos were at the 2-cell to 16-cell stages at 2–3 days of culture. Morulae and blastocysts were first observed at 4 and 5 days of culture after SCNT, respectively. Neither 1-cell to 4-cell embryos at 1–3 days of culture nor 5-cell to 16-cell embryos at 2–5 days of culture expressed EGFP (Table 2). In contrast, EGFP expression was first detectable in all the morulae tested at 4–

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Fig. 1.

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Fig. 2.

A: PCR analysis for the presence of the enhanced green fluorescent protein (EGFP) insert in G418-resistant miniature pig fetal fibroblast (MPFF) clones transfected with pOEIN. Genomic DNA was subjected to PCR using a primer set (EGFP5S/EGFP-5RV) corresponding to the middle region of EGFP in pOEIN transgene. M, 100-bp ladder markers; lanes 1–12, G418resistant clones; NC, non-transfected MPFFs; PC, pOEIN plasmid. B: Southern blot analysis for pOEIN-MPFF-#4 and pOEIN-MPFF-#5 cells (corresponding to lanes 4 and 5, respectively, in Fig. 2A). Lanes 4 and 5, pOEIN-MPFF-#4 and pOEIN-MPFF-#5 cells, respectively; C, non-transfected MPFFs; C1, C5, C10, C20 and C50, non-transfected MPFF DNA plus 1, 5, 10, 20 or 50 copies of pOEIN DNA, respectively.

Fig. 4.

Embryos reconstituted with pOEIN-MPFF-#4 cells after immunohistochemical staining using anti-Oct-3/4 antibodies under normal light (A–D) or ultraviolet (UV) light (E–H). The same embryos were also inspected for EGFP-derived fluorescence under UV light (I–L). A, E and I, a 3-cell embryo after culture for 2 days; B, F and J, a morula after culture for 5 days; C, G and K, a blastocyst after culture for 5 days; D, H and L, a blastocyst after culture for 7 days. Scale bar=50 μm.

Fig. 3.

pOEIN-MPFF-#4 cells and embryos reconstituted with them photographed under normal light (A–D) or ultraviolet (UV) light (E–H). As controls, parthenogenetically developed embryos were concomitantly observed under normal light (I and K) or UV light (J and L). A and E, pOEIN-MPFF-#4 cells immediately prior to somatic cell nuclear transfer; B, F, I and J, morulae after culture for 5 days; C, G, K and L, blastocysts after culture for 5 days; D and H, a blastocyst after culture for 7 days. Scale bars= 50 μm.

A: pOEIN plasmid used for transfection of miniature pig fetal fibroblasts. This plasmid has a pBluescript SK(–) backbone. The primer sets (EGFP-5S/EGFP-5RV and EGFP-7S/EGFP7RV) used for 1st PCR and nested PCR, respectively, are shown above the construct. The probe used for genomic Southern analysis is also shown below the construct as a solid bar. EGFP cDNA, enhanced green fluorescent protein cDNA; PGKp, mouse phosphoglycerate kinase promoter; neo, neomycin resistant gene. B: Mouse F9 and NIH3T3 cells transfected with pOEIN or pmaxGFP photographed under normal light or ultraviolet (UV) light. Arrows indicate EGFP expression in F9 cells transfected with pOEIN.

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Table 1. In vitro development of miniature pig somatic cell nuclear transfer embryos Duration of culture (days) 1 2 3 4 5 6 7 a

No. of trials

No. of embryos cultured

1-cell

2-cell to 4-cell

3 3 3 4 5 5 5

21 21 21 38 78 78 79

3 (14.3) 0 (0) 0 (0) 1 (2.6) 4 (5.1) 1 (1.3) 1 (1.3)

12 (57.1) 8 (38.1) 5 (23.8) 7 (18.4) 17 (21.8) 13 (16.7) 17 (21.5)

No. (%)a of embryos developed to 5-cell to 16-cell Morula 0 (0) 4 (19.0) 7 (33.3) 11 (28.9) 16 (20.5) 16 (20.5) 17 (21.5)

0 (0) 0 (0) 0 (0) 2 (5.3) 8 (10.3) 3 (3.8) 2 (2.5)

Blastocyst

Degenerated

0 (0) 0 (0) 0 (0) 0 (0) 6 (7.7) 14 (17.9) 9 (11.4)

6 (28.6) 9 (42.9) 9 (42.9) 17 (44.7) 27 (34.6) 31 (39.7) 33 (41.8)

Percentage per embryos cultured.

Table 2. Expression of enhanced green fluorescent protein (EGFP) in miniature pig somatic cell nuclear transfer embryos at different developmental stages 1-cell to 4-cell 1 2 3 Total No. of embryos examined 15 No. of embryos expressing EGFP 0 Percentageb of embryos expressing EGFP 0 a

8 0 0

5 0 0

28 0 0

Numbers under each stage indicate the duration of culture (days).

2 4 0 0

b

Developmental stage of embryosa 5-cell to 16-cell Morula 3 4 5 Total 4 5 6 7 0 0

11 0 0

16 0 0

38 0 0

2 2 100

8 8 100

3 3 100

Total

5

13 13 100

6 6 100

Blastocyst 6 7 14 4 28.6

9 0 0

Total 29 10 34.5

Percentage per embryos examined.

6 days of culture (IF=2.15; Fig. 3B and F). Although 10 of 20 (50.0%) blastocysts cultured for 5–6 days after SCNT expressed EGFP in both the ICM and TE (IF=2.85; Fig. 3C and G), EGFP expression was undetectable in any of the blastocysts tested at 7 days of culture (IF=1.10; Fig. 3D and H). On the other hand, none of the morulae and blastocysts developed from activated oocytes at 5 days of culture expressed EGFP (IF=1.05; Fig. 3I-L).

Expression of endogenous Oct-3/4 gene in SCNT embryos cultured in vitro When cleaved embryos at 2 days of culture, morulae at 5 days of culture and blastocysts at 5 or 7 days of culture were subjected to reaction with anti-Oct-3/4 antibodies after fixation and subsequent permeabilization, the reactivity peaked from the morula (IF=1.39) to blastocyst (IF=1.25) stages at 5 days of culture (Fig. 4B, C, F and G). As expected, cleaved embryos at 2 days of culture (IF=1.02) and blastocysts at 7 days of culture (IF=1.10) were negative for fluorescence (Fig. 4A, D, E and H). When the same embryos were inspected for EGFP-derived fluorescence, the fluorescence intensity also peaked from the morula to blastocyst stages at 5 days of culture (Fig. 4I-L). These results confirmed a correlation between the expression of Oct-3/4 promoter-directed transgene and that of endogenous Oct-3/4 gene.

Identification of the transgene in embryos To detect the presence of pOEIN transgenes in the blastocysts developed from SCNT embryos, their genomic DNA was first subjected to WGA. The WGA products were then subjected to PCR and a subsequent nested PCR. All the tested samples obtained from the 5 single blastocysts developed from embryos reconstituted with

Fig. 5.

PCR analysis for the presence of the enhanced green fluorescent protein (EGFP) insert in blastocysts developed from embryos reconstituted with pOEIN-MPFF-#4 cells. A single blastocyst was subjected to lysis and subsequent whole genome amplification (WGA). Similarly, genomic DNA from pOEINMPFF-#4 cells (positive control) or non-transfected miniature pig fetal fibroblasts (MPFFs; negative control) was subjected to WGA. PCR and subsequent nested PCR were performed using the WGA products to detect the EGFP element in pOEIN transgenes. M, 100-bp ladder markers; lanes 1–5, blastocysts developed from somatic cell nuclear transfer embryos; lanes 6– 10, blastocysts parthenogenetically developed; NC, nontransfected MPFFs; PC, pOEIN-MPFF-#4 cells.

pOEIN-MPFF-#4 cells exhibited a clear band of 400 bp corresponding to the EGFP sequence (Lanes 1–5 in Fig. 5). No band was visible when DNA from parthenogenetically developed blastocysts was similarly analyzed (Lanes 6–10 in Fig. 5). Isolation of the 400-bp band from gels and subsequent cloning into a TA cloning system revealed a high degree of fidelity concerning the amplified products; only one base was changed among the total 400-bp sequence (data not shown). These results indicate that pOEIN transgene was in fact transmitted to the progeny of the SCNT embryos.

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Discussion The results of the present study showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4–6 days after SCNT and that this noninvasive reporting system using Oct-3/4 promoter-driven EGFP transgene would be useful for evaluation of the reprogramming status of SCNT embryos. EGFP-derived fluorescence generated from embryos reconstituted with pOEIN-MPFF-#4 cells was observed at relatively limited stages, namely the morula to early blastocyst stages. This suggests that the Oct-3/4 expression level at stages before the morula stage and after the early blastocyst stage is low. The initial development of mammalian embryos is governed by gene transcripts and polypeptides produced by, and stored in, the oocytes during oogenesis [43]. However, following 1 to 3 cleavage divisions, expression of portions of the embryonic genome starts, and concomitantly, the maternally derived transcripts and proteins are gradually degraded [44–47]. This transition from maternal to embryonic gene expression is generally a gradual phenomenon. In pigs, global activation of the embryonic genome occurs at the 4cell to 8-cell stages [48]. The timing of embryonic genome activation of pig SCNT embryos is similar to that of in vivo-developed embryos [49]. Therefore, lack of Oct-3/4 expression in SCNT embryos at the 1-cell to 16-cell stages suggests that the genome of the pOEIN-MPFF-#4 cells transferred into the recipient oocytes had not been fully activated or reprogrammed. Lee et al. [29] demonstrated using RT-PCR that Oct-3/4 transcripts are abundantly detected in SCNT embryos derived from MPFFs at the morula and blastocyst stages. In addition, the relative amount of Oct-3/4 mRNA is significantly reduced in hatched blastocysts developed from SCNT embryos compared with that of hatched blastocysts developed in vivo or produced via in vitro fertilization. Our present results appear to be consistent with the results of Lee et al. [29]. In contrast to the situation in mice [19, 20], expression of Oct-3/ 4 in the TEs of pig and cattle embryos at the pre- and peri-hatching blastocyst stages does not appear to be down-regulated [26–28]. During the immediate post-hatching period, however, Oct-3/4 expression becomes restricted to the ICM and epiblast [28, 50–52]. In the present study, localization of EGFP expression in blastocysts developed from embryos reconstituted with pOEIN-MPFF-#4 cells was not only confined to the ICM but was even seen in the TE. This finding is consistent with the previous reports. Furthermore, immunohistochemical staining using anti-Oct-3/4 antibodies revealed that the EGFP expression pattern seen in the embryos reconstituted with pOEIN-MPFF-#4 cells was strictly correlated with that of the endogenous Oct-3/4 gene. Although we have succeeded in producing cloned piglets using the procedures of SCNT described in the present study, the efficiency of cloning, which can be assessed as the number of delivered offspring among the number of embryos transferred to recipient females, was only 1.4% [38]. The results of the present study suggest that the above-mentioned low cloning efficiency may be related to lack of Oct-3/4 expression in the SCNT embryos at the late blastocyst stage. Late blastocysts developed in vivo or produced via in vitro fertilization express Oct-3/4 at high levels [29].

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Oct-3/4 is known to either repress or activate target gene expression [53–57] and regulate the expression of multiple genes downstream [58]. Therefore, the lack of Oct-3/4 expression in SCNT embryos at the later stages of preimplantation development may inhibit their normal development to term. In mice, if the ICMs of blastocysts contain inappropriate levels of Oct-3/4, the capacity of these cells to form embryonic lineages is reduced [31]. In this context, development of procedures to increase Oct-3/4 expression at those stages may be critical for improvement of cloning efficiency. In other words, the present system using pOEIN transfectants would be useful for screening drugs supporting in vivo development of SCNT embryos. With this system, we have recently demonstrated that valproic acid, a histone deacetylase inhibitor, is beneficial for prolonged expression of Oct-3/4 as well as for increasing the blastocyst formation rates of SCNT embryos [59]. In addition, our system is helpful for establishment of ES cell lines by long-term serial passage of ICM-derived cells, since pluripotent-state ES cells can be simply and noninvasively monitored under a fluorescent microscope. On the other hand, although a high proportion of mouse blastocysts developed from embryos reconstituted with PGCs have normal Oct-3/4 expression, their post-implantation development is extremely poor [31]. This suggests that there are considerable additional requirements that need to be fulfilled for development of SCNT embryos, other than reprogramming of Oct-3/4. Therefore, it may be necessary to examine other genes regarding both the levels of expression and spatial distribution after establishment of procedures to increase Oct-3/4 expression at the late blastocyst stage. Initially, to examine whether the exogenous pOEIN transgene is present in blastocysts developed from SCNT embryos, a single blastocyst was subjected to direct PCR using blastocyst lysate and subsequent nested PCR in the present study. However, in this case, no clear band was visible in any of the tested samples (data not shown). We ascribed this failure to the very low amount of blastocyst genomic DNA itself or to use of an inappropriate primer set for sensitive detection of a target gene. Thus, we considered amplification of the genomic DNA of the blastocysts by a WGA technique prior to PCR. When each blastocyst lysate was subjected to rolling circular amplification (RCA) and a portion of the RCA products was subjected to PCR and a subsequent nested PCR, all the tested samples exhibited a clear band. Therefore, we consider RCA prior to PCR to be useful for detection of a target gene in genomic DNA obtained from a single embryo. In conclusion, we developed a simple and noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig SCNT embryos. This system would be useful for exploration of optimal SCNT conditions that lead to improvement of cloning efficiency.

Acknowledgments We thank Dr K Ohbo (School of Medicine, Yokohama City University, Yokohama, Japan) for providing the pGOF-18 plasmid. We also express gratitude to the staff of the Kagoshima City Meat Inspection Office and Meat Center Kagoshima (Kagoshima, Japan)

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for supplying pig ovaries. The present study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 17100007 and 20580311 to MY and 19580328 to KM).

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