Production of Transgenic Wuzhishan Inbred Miniature Pigs Carrying Green Fluorescent Protein by Somatic Cell Nuclear Transfer

In this study, based on somatic cell nuclear transfer (SCNT), we used the ear fibroblasts of miniature pigs transfected with enhanced green fluorescent protein (EGFP) as the donor cells and transplanted reconstructed embryos (at the 1–8 cell stage) into the oviducts of three sexually mature Wuzhishan miniature pigs that were naturally estrus. One recipient successfully birthed two stillborn and two live piglets after 116 days of pregnancy. We investigated the presence of the foreign gene in the skin tissues of the piglets and tested 13 microsatellite polymorphisms in the recipient pigs, cloned offspring and donor cells. The results indicated that two cloned offspring expressed EGFP and all four cloned offspring had the same polymorphisms at all 13 microsatellites as the donor cell. However, the microsatellites varied between the cloned offspring and the recipient pigs, indicating the absence of a blood relationship between the two. The study therefore establishes a system to obtain transgenic Wuzhishan inbred miniature pigs by SCNT, thereby providing a basis to study the pathologic mechanisms of human disease and producing animal models for human


INTRODUCTION
Miniature pigs are considered to represent an attractive species for use as experimental models for biomedical research due to the many characteristics (including anatomy and physiology) that are similar to humans and also because their small body size makes them easier to handle and cheaper to maintain compared with larger domestic pigs [1].The Wuzhishan inbred miniature pig is a Chinese variety that has the highest inbreeding coefficient and is a candidate experimental model.
The enhanced green fluorescent protein (EGFP) is derived from a kind of jellyfish and is commonly used as a marker because it is easy to visualize under UV light [2][3][4][5][6].EGFP makes it easy to detect transgenic animals; therefore it has been widely used as a marker in transgenic studies.Although the production of a transgenic EGFP domestic pig by somatic cell nuclear transfer (SCNT) has been reported by several research groups [7][8][9][10][11], there is no information available on the production of transgenic EGFP Wuzhishan inbred miniature pig clones using donor cells from Wuzhishan miniature pigs.
In our study, we selected EGFP as a target gene, and detected in vitro and in vivo development and EGFP expression in SCNT embryos that were derived from EGFP-positive cells.Finally, we showed in vivo developmental competence of SCNT Wuzhishan miniature pig embryos to term.

MATERIALS AND METHODS Plasmids, chemicals and animals
The plasmid pFB-EGFP was offered by Professor Jin from the Academy of Military Medical Sciences of the PLA, China.All chemicals used in this study were purchased from Sigma Chemical Company (Sigma-Aldrich, China), unless otherwise indicated.The laboratory animals were purchased from the Agricultural Science and Technology Institute of Yanbian, China.This research was carried out in accordance with the Ethics Committee of Yanbian University.

Isolation and culture of porcine somatic cells
The miniature pig fibroblasts were obtained from the ears of Wuzhishan miniature pigs.Ear tissues were cut into small pieces and cultured in a mixture of Dulbecco modified Eagle medium (DMEM) and Ham F-12 medium (Gibco, USA) supplemented with 10% (v:v) fetal calf serum (FCS) in 5% CO 2 in air at 38°C.After reaching confluence, cells were passaged several times.Cells between passages 4 and 8 were used as donors for nuclear transfer.The cells were allowed to grow to confluence and cultured for an additional 5-6 days without a change of medium.A single cell suspension was prepared by standard trypsinization immediately prior to nuclear transfer.

Transfection of pFB-EGFP into ear fibroblasts
The day before transfection, confluent ear fibroblasts (at passage 3 to 5) were trypsinized, counted and plated onto 35 mm culture dishes to reach 80% confluency on the day of transfection.One microliter (1 μl) of the pFB-EGFP plasmid and 8 ml of FuGENE ® HD (Roche Diagnostics, Indianapolis, IN, USA) were diluted with 91 ml of serum-free DMEM.After 15 min of incubation at room temperature, 100 ml of the DNAmedium mixture was added to 2 ml of cell culture medium.The cells were cultured for 2-3 days until confluency and passaged once to achieve stable integration of the transgene into chromosomes before use.For serum starvation, cells were cultured for 3 days in DMEM supplemented with 0.5% FCS prior to SCNT and individual cells were retrieved from the monolayer by trypsinization for 30 s.

Nuclear transfer
Nuclear transfer was carried out as described by Yin et al. [12].Briefly, matured eggs with the first polar body were cultured in medium supplemented with 0.4 mg/ml demecolcine (Sigma) and 0.05 mol/l sucrose for 1 h.Sucrose was used to enlarge the perivitelline space of the eggs.Treated eggs with a protruding membrane were moved to medium supplemented with 5 mg/ml cytochalasin B and 0.4 mg/ml demecolcine and the protrusion was removed with a beveled pipette.A single donor cell was injected into the perivitelline space of each egg and electrically fused using two direct current pulses of 150 V/mm for 50 ms in 0.28 mol/l mannitol supplemented with 0.1 mM MgSO 4 and 0.01% polyvinyl alcohol (Sigma).Fused eggs were cultured in medium with 0.4 mg/ml colcemid for 1 h before parthenogenetic activation, and then cultured in 5 mg/ml of cytochalasin B-supplemented medium for 4 h.The reconstructed oocytes were activated by two direct current pulses of 100 V/mm for 20 ms in 0.28 mol/l mannitol supplemented with 0.1 mmol/l MgSO 4 and 0.05 mmol/l CaCl 2 .Activated eggs were cultured in the medium for 6 days in an atmosphere of 5% CO 2 and 95% air at 39°C.

Embryo transfer
Sexually mature Wuzhishan miniature pigs (aged 2-3 years with a body weight between 30-50 kg) were used as the recipients for the embryo transfer.Eggs that had undergone nuclear transfer were surgically transferred into the oviducts of Wuzhishan miniature pigs at natural estrus.

PCR
The screening procedure for the presence of the EGFP transgene involved the isolation of genomic DNA from porcine adult ear skin-derived fibroblast cells and the amplification of a PCR fragment encompassing the promoter-gene junction.The PCR product of 728 bp was amplified with forward primers (5′-TGA ACC GCA TCG AGC TGA AGG G-3′) and reverse primers (5′-TCC AGC AGG ACC ATG TGA TCG C-3′).The PCR reaction mixture contained: 2 μl of genomic DNA, 25 mM MgCl 2 , 1 μl of 10 mM deoxynucleoside triphosphates, 1 μl of each primer at 20 μM and 0.5 μl of 5 U of Taq DNA polymerase (Takara Bio Inc., Shiga, Japan) in a final volume of 25 μl.The PCR reaction was performed using the following conditions: denaturation at 94°C for 45 s; annealing at 56°C for 30 s; synthesis at 72°C for 30 s; for 30 cycles.The PCR products were fractionated on a 1% agarose gel.

Microsatellite analysis
Parentage analysis was performed on the piglets obtained by nuclear transfer and the surrogate recipient females to confirm the identity of the donor cells used for nuclear transfer.DNA was extracted from ear punches or tail clippings obtained from each newborn piglet, the recipient females and donor cells.Five porcine DNA microsatellite markers (SW936, SW951, SW787, S00090, S0026, SW122, SW857, S0005, SW72, S0155, S0225, SW24, SW632) were used to confirm that the cloned piglets shared a genetic background with the donor cells used for nuclear transfer.

Statistical analysis
For proportional data, differences between groups were analyzed using the Chi-square test.For blastocyst cell number data, differences between groups were determined using the student's t-test.The level of significance was set at P < 0.05.

Transfection of somatic cells and screening
Recombinant plasmid pFB-EGFP transfected fibroblasts WMP of pig ear after 72h, to a final concentration of 200 μg/mL G418, 10% fetal bovine serum in DMEM selection medium filter, medium was changed every other day, 10d, can be observed recombinant virus to uninfected cells all died, and the cells infected with recombinant virus screening of 3 from the beginning has gradually formed a G418-resistant cell clones and Can be observed under a fluorescence microscope bright green fluorescence (Figure 1).

In vitro development of cloned miniature pig embryos reconstructed with non-transfected or transfected ear fibroblasts
The production efficiency of nuclear transfer embryos reconstructed using Wuzhishan miniature pig ear fibroblasts as donor cells and the in vitro development of nuclear transfer embryos is summarized in Table 1.No significant differences were observed in the fusion rates and cleavage rates in non-trasfected or transfected ear fibroblasts (84.38%, 72.63% and 81.42%, 74.50%).EGFP gene transfection did not affect the rate of embryo development to blastocyst stage or the total cell numbers in blastocysts.Blastocysts derived by SCNT of miniature pig ear fibroblasts transfected with EGFP were clearly observed under a fluorescent microscope (Figure 2).

Development of cloned miniature pig embryos after nuclear transfer
The results of transferring the cloned embryos into recipient pigs are shown in Table 2. Embryos reconstructed using miniature pig ear fibroblasts transfected with EGFP were transferred into three recipient miniature pigs.Following the transfer of cloned embryos carrying a copy of the EGFP gene, two recipients become pregnant, and while one miscarried (day 57), the other delivered two live piglets and two stillborn piglets.The birth weights of the live offspring were 366 g and 573 g whereas the two stillborn piglets weighed 247 g and 313 g.PCR showed the presence of the EGFP gene in genomic DNA extracted from the tissue of two of the four cloned pigs (Figure 3).In addition, fluorescence was observed in the hooves of two cloned piglets under a fluorescence microscope.A microsatellite analysis was conducted by sending samples obtained from the cloned miniature pigs, donor cells and recipients to a laboratory that specializes in porcine parentage verification (Molecular laboratory, Gyeongsang University, South Korea), and the results confirmed that the piglets were clones of the donor cells(Table 3).

DISCUSSION
This study showed that porcine nuclear transfer embryos reconstructed with ear fibroblasts transfected using the FuGENE ® HD transfection reagent had higher developmental competence.After transfer, the reconstructed embryos developed to full term, leading to the birth of live-born offspring.These results indicate that nuclear transfer from somatic cells transfected with EGFP provides a non-invasive method for the production of transgenic porcine embryos.
In previous study, we compared the efficiencies of two transfection reagents to transfect porcine fetal fibroblasts.A clear difference in transfection efficiency between FuGENE ® HD and Lipofectamine 2000 was apparent.Transfection with FuGENE ® HD seems to deliver DNA into porcine fetal fibroblasts by an order of magnitude more efficiently.When transgene expression was assessed, EGFP protein was detected from 1-2 h post-transfection to the blastocyst stage in the reconstructed embryos.Previous results suggest that after the insertion of the transfected donor cells into cytoplasts, EGFP protein is dispersed into the cytoplasm of oocytes and the EGFP gene also produces EGFP in the cytoplasm [13].In the present study, 100% of blastocysts transfected with FuGENE ® HD expressed EGFP protein when assessed under UV.However, when these transfected somatic cells were used as donors, only 50% of the cloned transgenic miniature pigs carried the EGFP gene when detected by PCR.In addition, fluorescence was detected in the hooves of only 50% of the cloned offspring under a 365 nM portable UV lamp.This may be caused by insufficient G-418 selection of EGFP transfected donor cell lines or the extinction of the foreign gene.
In the present study, oocytes from the common domestic pig, collected from an abattoir, were used as the recipient cytoplasm for nuclei from Wuzhishan miniature pigs; however, the effect of utilizing common domestic pig oocytes on the physiological characteristics of cloned Wuzhishan miniature pigs has not been clarified.The average birth weight of the two live cloned piglets produced by recipient Wuzhishan miniature pigs was 469 g, which is comparable to the average birth weight of piglets produced by naturally bred Wuzhishan miniature pigs.
We demonstrated the in vivo developmental competence of SCNT embryos produced using a promising SCNT protocol established in this study.We used Wuzhishan miniature pigs as the surrogate recipients.Miniature pigs are considered to have the advantage of easy handling compared with domestic pigs.Their small body size enables space saving and provides potential benefits for the control of specific pathogens in the breeding facilities [14].In previous studies, SCNT embryos reconstructed using donor cells from miniature pigs have been transferred to domestic pigs used as surrogate mothers [15,16] .Hoshino et al. have shown that after the transfer of SCNT embryos into Göttingen minipigs or Meishan pigs, only the Meishan pigs became pregnant.The authors conjectured that oocytes derived from domestic pigs may not fit into the uterus of a miniature pig [17].In the present study, however, SCNT embryos reconstructed fetal fibroblasts of miniature pigs were transferred to surrogate Wuzhishan miniature pigs, and developed to full term.To our knowledge, this is the first report of producing a SCNT miniature piglet by using surrogate Wuzhishan miniature pigs.
In summary, the use of cationic liposomes as a reagent to introduce foreign DNA into porcine ear somatic cells and the use of transfected ear fibroblasts as donor cells offers an efficient and practical method of producing transgenic pigs.Furthermore, Wuzhishan miniature pigs can be used as surrogate mothers.

Fig. 1 .
Fig. 1.G418 screening of the transgenic fetus fibroblasts at 10 days (×40).(A) The cells under a light microscope; (B) the cells under a fluorescence microscope.

Table 1 :
In vitro development of cloned embryos reconstructed with transfected or non-transfected donor cells in miniature pigs.

Table 2 :
Production of cloned miniature pigs by nuclear transfer of ear fibroblast cells transfected with the EGFP gene.