CLONING AND STEM CELLSVolume 9, Number 4, 2007© Mary Ann Liebert, Inc.DOI: 10.1089/clo.2007.0028

Brief Communication

Transgenesis and Nuclear Transfer Using PorcineEmbryonic Germ Cells

KWANG SUNG AHN, JI YOUNG WON, SOON YOUNG HEO, JEE HYUN KANG, HONG SEOK YANG, and HOSUP SHIM

ABSTRACT

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordialgerm cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differen-tiation have been established. Because EG cells can be cultured indefinitely in an undiffer-entiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) thansomatic cells that have limited lifespan in primary culture. Use of EG cells could be particu-larly advantageous to provide an inexhaustible source of transgenic cells for NT. In this studythe efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were com-pared. The rate of development to the blastocyst stage was significantly higher in EG cell NTthan somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can beused for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced intoporcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts(29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. Theresults obtained from the present study suggest that EG cell NT may have advantages oversomatic cell NT, and transgenic pigs may be produced using EG cells.

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INTRODUCTION

CLONING BY SOMATIC CELL NUCLEAR TRANSFER

(NT) has been achieved in several mam-malian species including sheep (Wilmut et al.,1997), cattle (Cibelli et al., 1998), goat (Baguisi etal., 1999), mice (Wakayama et al., 1998), and pigs(Polejaeva et al., 2000). However, the rates of gen-erating live offspring from NT embryos havebeen disappointingly low. The reasons behind thefailures maintaining pregnancy are not yet fullyunderstood. They may originate from the recipi-ent cytoplast (e.g., insufficient activation, lack of

reprogramming factors, reduced viability owingto micromanipulation), or from the DNA of thedonor cell (e.g., inappropriate nuclear status, in-complete preprogramming, chromosomal abnor-malities, damaged DNA), or both. One of the fac-tors to determine the efficacy of NT might be thetype of donor cells. A comparison of efficacyamong donor cells of different types includingundifferentiated stem cells would be of interestbecause a less differentiated cell type may sup-port greater development of NT embryos com-pared with terminally differentiated cell types(Faast et al., 2006).

Department of Physiology, Dankook University School of Medicine, Cheonan, Chungnam, Korea.

Primordial germ cells (PGC) are embryoniccells that migrate from the root of the allantois tothe genital ridge, where they ultimately give riseto gametes. Cells having morphological, bio-chemical, immunological, and developmentalproperties in common with embryonic stem (ES)cells, including pluripotency and the capacity tocontribute to the germ line of chimeras, have beenisolated from murine PGC (Labosky et al., 1994;Matsui et al., 1992; Resnick et al., 1992). ThesePGC-derived stem cells have been called embry-onic germ (EG) cells to distinguish them from EScells. Undifferentiated porcine EG cell linesdemonstrating capacities of both in vitro and invivo differentiation have been established (Shimet al., 1997). Unlike somatic cells that have lim-ited lifespan, EG cells can be cultured indefinitelyin an undifferentiated state. Providing an abun-dance of pluripotent stem cells that can be ge-netically manipulated by conventional recombi-nant DNA techniques may enable stable geneticmutations to be established and maintained. Ifthese cells are used for a source of karyoplasts inNT, it would be particularly advantageous inproducing transgenic animals. Hence, in thisstudy porcine EG cells were tested for nucleardonor cells to improve the efficiency in the pro-duction of NT embryos.

The green fluorescent protein (GFP) gene hasbeen successfully used as a marker gene fortransgenesis in embryos (Takada et al., 1997). Inpigs, the GFP gene was expressed in NT clonedembryos derived from transfected fetal fibroblastcells (Park et al., 2001a, 2001b). Recently a stabletransfection of the GFP gene into porcine EG cellshas been reported (Rui et al., 2006). In the pres-ent study, the procedure was developed for moreimproved and stable transfection of porcine EGcells using GFP transgene construct. Further-more, we tested the effects of transgenesis in EGcells on subsequent development of recon-structed oocytes. The results of the present studymay contribute to overcoming current problemsof low efficiency of transgenesis and NT using so-matic cells.

MATERIALS AND METHODS

Animal ethics

All procedures in this study were carried outin accordance with the Code of Practice for the

Care and Use of Animals for Scientific Purposesapproved by Animal Ethics Committee, DankookUniversity School of Medicine.

In vitro maturation of porcine oocytes

Porcine oocytes were matured in vitro by themethod modified from Hyun et al. (2003). Briefly,ovaries were collected from prepubertal gilts at alocal slaughterhouse and transported to labora-tory in a warm box (25 to 30°C) within 2 h. Fol-licular fluid and cumulus–oocytes complexes(COC) from follicles of 5 to 6 mm in diameterwere aspirated using an 18-gauge needle attachedto 5-mL disposable syringe. Compact COC wereselected and washed six times in HEPES-bufferedtissue culture medium (TCM)-199 (Gibco BRL,Gaithersburg, MD). The in vitro maturation (IVM)medium was modified TCM-199 (Gibco BRL)supplemented with 10 ng/mL epidermal growthfactor (Sigma, St. Louis, MO), 10 IU/mL pregnantmare serum gonadotropin (PMSG; Intervet,Boxmeer, The Netherlands), 10 IU/mL humanchorionic gonadotropin (hCG; Intervet) and 10%(v/v) porcine follicular fluid. A group of 50 COCwas cultured in 500 �L of IVM medium at 39°Cin a humidified atmosphere of 5% CO2 and 95%air. After culturing for 22 h, COC were trans-ferred to PMSG- and hCG-free IVM medium andcultured for another 20 h. At the end of the mat-uration, oocytes were freed from cumulus cellsby repeated pipetting in the IVM medium con-taining 0.5 mg/mL hyaluronidase (Sigma) for 1min.

Preparation of porcine fetal fibroblasts

Fibroblasts were isolated from pig fetuses onday 23 of gestation. Briefly, fetuses were washedthree times with Ca2�- and Mg2�-free phosphate-buffered saline (PBS; Gibco BRL). The heads andinternal organs were removed using iris scissorsand forceps. The remnants were washed twice inDulbecco’s PBS (DPBS), minced with a surgicalblade on a 100-mm Petri dish. Cells were disso-ciated from the tissues in 0.25% (v/v) trypsin-EDTA (Gibco BRL) for 5 min at 39°C. After cen-trifuging cell suspension three times at 800 � gfor 10 min, pellets were subsequently seeded onto100-mm plastic culture dishes (Falcon, FranklinLakes, NJ), and cultured for 6 to 8 days in Dul-becco’s modified Eagle medium (DMEM; GibcoBRL) supplemented with 10% (v/v) fetal bovineserum (FBS; Gibco BRL), 1 mM L-glutamine

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(Gibco BRL), 100 units/mL penicillin (Gibco BRL)and 0.5 mg/mL streptomycin (Gibco BRL) in ahumidified atmosphere of 5% CO2 in 95% air. Af-ter removal of unattached clumps of cells, at-tached cells were further cultured until confluent,subcultured at intervals of 5 to 7 days by tryp-sinization for 5 min using 0.25% trypsin–EDTA,and stored after two passages in freezing mediumin liquid nitrogen at �196°C. The freezingmedium consisted of 80% (v/v) DMEM, 10%(v/v) dimethyl sulfoxide (Sigma) and 10% (v/v)FBS. After thawing, cells were cultured in DMEMsupplemented with 10% FBS until approximately80% confluency and used for NT experiment.

Isolation, culture, and transfection of porcine EG cells

Porcine EG cells were isolated from PGC of day23 embryos collected from Hampshire � York-shire crossbred gilts as described previously(Shim et al., 1997). Briefly, the dorsal mesenterywas removed from the embryos and placed in0.02% EDTA solution for 20 min. Primordial germcells were released from the dorsal mesentery bygentle pressing and pricking the tissue using fineforceps and collected by centrifugation at 800 �g for 5 min. Harvested PGC were cultured inDMEM containing 15% ES-qualified FBS (GibcoBRL), 1 mM L-glutamine, 0.1 M MEM nonessen-tial amino acids, 10 �M 2-mercaptoethanol, 100units/mL penicillin, 0.5 mg/mL streptomycinand 1000 units/mL murine leukaemia inhibitoryfactor (Chemicon, Temecula, CA) (designatedPGC culture medium), on inactivated STO feedercells prepared by the treatment of 10 �g/mL mitomycin C (Sigma) for 2 h. Approximately30,000 PGC were seeded per well of a 96-wellplate (Falcon) containing feeder cells. ResultingEG cell colonies from PGC culture were disag-gregated by incubation in 0.25% trypsin–EDTAfor 10 to 15 min and subcultured onto fresh feedercells in a four-well multidish (Nunclon, Roskilde,Denmark) approximately every 5 to 7 days. Allcultures were maintained at 39¯C in 5% CO2, 95%air with culture medium changed every otherday.

Enhanced green fluorescent protein (EGFP)vector pEGFP-N1 (Clontech, Palo Alto, CA) con-sisting of a selectable neor gene as well as theEGFP gene attached to the cytomegalovirus(CMV) promoter was amplified in DH5� compe-tent cells, and plasmid DNA was isolated using

Plasmid Maxiprep kit (Promega, Madison, WI)according to the manufacturer’s protocol. To in-crease an efficiency of transgene integration intothe genome of EG cells, the vector was linearizedby EcoO 109 restriction, resulting in a 4.7-kb DNAfragment containing the genes encoding EGFPand neor under the regulation of separate pro-moters.

The mixture of trypsinized EG and feeder cellswere cultured for 15 min onto 0.1% gelatin-coatedplate until fibroblasts were attached on the dishwhile the most of EG cells were floating. Purepopulation of EG cells was microscopically con-firmed based on their size because individual EGcells were 5 to 15 �m in diameter, approximatelya third the size of a STO feeder cell (Shim et al.,1997). Then, the cells in the supernatant were col-lected and washed by centrifugation at 800 � gfor 5 min, and transferred onto 0.1% gelatin-coated plate. Continuously EG cells were grownin PGC culture medium in a humidified atmo-sphere of 5% CO2 in 95% air. Feeder-free porcineEG cells were prepared separately from the 15th,16th, and 18th passages of EG cells. After four tofive additional passages of feeder-free EG cells,no morphological differences were noticedamong three feeder-free cell lines, and one feeder-free cell line was randomly selected for transfec-tion to establish EGFP-transfected cell lines(EGFP–PEGC). Transfection was performed us-ing Effectene (Qiagen, Valencia, CA) according tothe manufacture’s protocol. Twenty-four hoursafter transfection, the cells were selected withmedium containing 500 �g/mL of G418 (GibcoBRL) for 7 to 10 days. After antibiotic selection,cells were transferred on fresh gelatinized dishesand cultured at 39°C in a humidified atmospherecontaining 5% CO2 in 95% air until use.

Nuclear transfer

At 42 h after the onset of IVM, oocytes wereenucleated with a 20-�m (internal diameter) glasspipette by aspirating the first polar body and thesecond metaphase plate with a small volume ofsurrounding cytoplasm in HEPES-buffered TCM-199 supplemented with 0.4% bovine serum albu-min (BSA; Sigma) and 7.5 mg/mL cytochalasin B(Sigma). After the enucleation, oocytes werestained with 5 mg/mL bisbenzimide (Hoechst33342; Sigma) for 5 min and observed under aninverted microscope equipped with epifluores-cence. Oocytes containing DNA materials were

PORCINE EG CELL NUCLEAR TRANSFER 463

excluded from the subsequent experiments. Oneporcine EG and one fibroblast cell line (i.e., froma single donor embryo each) were used for donorcells in NT experiment. Either transfected or non-transfected nuclear donor cells were trypsinizedinto single cells, selected under an inverted mi-croscope equipped with a GFP filter and trans-ferred into the perivitelline space of enucleatedoocytes. The resulting couplets were equilibratedfor 1 min in 0.3 M mannitol solution containing0.5 mM HEPES, 0.05 mM CaCl2, and 0.1 mMMgCl2 in a chamber containing two electrodes.Then, couplets were fused with double DC pulseof 1.5 kV/cm for 40 �sec using BTX Electro-CellManipulator 2001 (Gentronics, San Diego, CA).Following the electrical stimulation, recon-structed oocytes were washed three times withNCSU 23 supplemented with 4 mg/mL fattyacid-free BSA (Sigma) and cultured in the samemedium containing 7.5 mg/mL cytochalasin Bfor 3 h to suppress extrusion of the second polarbody. Next, reconstructed oocytes were culturedfor 4 days in NCSU 23 containing 4 mg/mL fattyacid-free BSA and transferred to NCSU 23 con-taining 10% FBS and cultured for another 3 days.All NT embryos were cultured at 39°C in a hu-midified atmosphere containing 5% CO2 in 95%air, and the expression of the transgene was mon-itored under fluorescent microscope. In addition,the rate of in vitro development of NT embryosderived from EGFP–PEGC was compared tothose from nontransfected counterpart.

Statistical analysis

In the comparison of two different types ofdonor cells (fibroblasts vs. EG cells) both celltypes were tested in each replicate. Transgenicand nontransgenic cells were tested in the sameway. At least three replicates were conducted foreach experiment.

Data on the rates of fusion, cleavage, and sub-sequent development to the blastocyst stage weresubjected to Student’s t-test. Differences of p �0.05 were considered to be significant.

RESULTS

Development of somatic and EG cell nucleartransfer embryos

In vitro development of embryos after NT of fi-broblast and EG cells was shown in Table 1. Be-tween two different donor cells used for NT inthis study, no differences were observed in therate of fusion and cleavage. However, the rate ofblastocyst development from EG cell NT was18.1% (94 blastocysts from 518 fused oocytes) andsignificantly higher than 14.4% in blastocyst de-velopment (72 blastocysts from 501 fusedoocytes) from somatic cell NT.

Transgenesis of porcine EG cells

Transfection of feeder-free porcine EG cells us-ing polycationonic lipid and subsequent antibi-otic selection of cells carrying transgene resultedin expression of the EGFP gene based on obser-vation under fluorescent microscope. Subcultureof such selected cells produced colonies consist-ing of EG cells emitting green fluorescence asshown in Figure 1.

Development of nuclear transfer embryos fromtransgenic EG cells

Nuclear transfer embryos from transfected EGcells expressed EGFP under fluorescent micro-scope. Transgene expression was observedthroughout preimplantation development fromas early as late one-cell stage (Fig. 2). Table 2 rep-resents the comparison of in vitro development

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TABLE 1. IN VITRO DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

Nuclear donor No. of No. (%) ofcells oocytes fused oocytesa Two cellb Blastocystb

Fibroblasts 885 501 (56.6) 287 (57.3) 72 (14.4)c

EG cells 794 518 (65.2) 289 (55.8) 94 (18.1)d

aCalculated from the number of oocytes.bCalculated from the number of fused oocytes.c,dp � 0.05.

No. (%) of embryos developed to

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FIG. 1. Transgenesis of porcine EG cells. (A) Porcine EG cell colony in culture (200�). (B) The same colony in panelA under fluorescent microscope (200�). (C) Feeder-free porcine EG cell colonies (100�). (D) The same colonies inpanel C under fluorescent microscope (100�).

FIG. 2. Expression of transgene in nuclear transfer embryos from EGFP-transfected EG cells. (A) Two-cell stage em-bryos at 26 h postnuclear transfer (100�). (B) The same embryos as in panel A under fluorescence microscope (100�).(C) Blastocyst-stage embryo at 146 h postnuclear transfer (200�). (D) The same embryo as in panel C under fluores-cence microscope (200�).

between NT embryos from transfected and non-transfected EG cells. The rate of blastocyst devel-opment in NT embryos from transfected EG cellswas 21.2% (29 of 137) and comparable to thatfrom nontransfected control (18.4%, 25 of 136).This result suggests that the transgenesis of EGcells did not significantly affect preimplantationdevleopment of NT embryos.

DISCUSSION

Although somatic cell NT has been a widelyused procedure to generate cloned offspring inmammals, use of undifferentiated stem cells fornuclear donor may be advantageous becausesuch cells could be more easily reprogrammable.In mice, for instance, oocytes reconstructed fromES cells gave rise to an increase in the number ofviable offspring compared with those from so-matic cells (Rideout et al., 2000; Wakayama et al.,1999). However, similar studies have not beenperformed in domestic animals perhaps due tolimited availability of ES cells. Instead, adult stemcells such as mesenchymal stem cells isolatedfrom porcine bone marrow resulted in the ratesof preimplantation development comparable to(Bosch et al., 2006; Colleoni et al., 2005; Faast etal., 2006) or better than (Jin et al., 2007) their so-matic cell counterpart. In addition, embryoscloned from porcine skin-originated sphere stemcells from fetal skin showed enhanced preim-plantation development compared with fibro-blast cloned embryos, which is indicated by anincreased rate of blastocyst development and ahigher total cell number in Day 7 blastocysts (Zhuet al., 2004). These results imply that stem cellpopulation may be used for alternative source ofnuclear donor cells for NT.

We have previously reported an isolation of EGcells, stem cells derived from PGC (Shim et al.,

1997). These EG cells that were pluripotent bothin vitro and in vivo were tested for their appro-priateness for karyoplasts in NT experiment inthe present study. The result shown in Table 1suggests that the type of nuclear donor cells iscritical for determining developmental compe-tence. Stem cell populations, such as EG cells, arehighly undifferentiated cells compared withother cell types retrieved from adult tissue, andhave a greater potential as donor cells than fetalfibroblast in achieving enhanced production ofcloned porcine embryos. The superiority of EGcells as karyoplasts for NT demonstrated in thisstudy suggests that undifferentiated stem cellsare more amenable to reprogramming after re-construction than differentiated somatic cells.

Because undifferentiated stem cells such as ESor EG cells can be maintained indefinitely in cul-ture, use of such cells for transgenesis may facil-itate ease of gene transfer and subsequent selec-tion of transgenic cells. The current problem ofusing terminally differentiated somatic cells isthat they tend to become senescent before suffi-cient rounds of gene transfer and/or gene tar-geting followed by antibiotic selection. This maybe overcome by isolation and use of cell lines thatare capable of transfection and long-term culture.Porcine EG cells may possess such characteristics.Introduction of the exogenous EGFP gene intoporcine EG cells has recently been reported (Ruiet al., 2006). However, in this report performingtransfection of EG cells on the feeder layer thetransgenesis was inconsistent depending on lipo-fection methods and EG cell lines used. In thepresent study, porcine EG cells in feeder-free cul-ture were transfected with the transgene EGFP.Transfected EG cells selected by antibiotics treat-ment maintained stable expression of EGFP inculture for several months without an overt signof differentiation (Fig. 1). This would be particu-larly advantageous in maintaining nuclear donor

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TABLE 2. IN VITRO DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

FROM TRANSFECTED AND NONTRANSFECTED EG CELLS

Nuclsear donor No. of No. (%) ofcells oocytes fused oocytesa Two cellb Blastocystb

Transfected 237 137 (57.8) 76 (55.5) 29 (21.2)Nontransfected 210 136 (64.8) 79 (58.1) 25 (18.4)

aCalculated from the number of oocytes.bCalculated from the number of fused oocytes.

No. (%) of embryos developed to

cells carrying a transgene. If combined with NTtechnique, EG cells may potentially be useful forgenetic manipulation in pigs.

Furthermore, chromosomal stability of porcineEG cells as previously reported (Shim et al., 1997)may yield consistent results in NT compared withsomatic cells such as fetal fibroblasts that oftenexhibit chromosomal abnormality in long-termculture (Mir et al., 2003). There was no differenceof developmental rate between reconstituted em-bryos derived from transgenic EG cells and non-transgenic control (Table 2), suggesting thattransgenesis of EG cells might not affect the sub-sequent development of embryos after NT. Theexpression of the EGFP gene in the embryos wasobserved by fluorescence microscope as the earlyas late one-cell stage far before the embryonic ge-nome activation begins (Fig. 2). This may repre-sent episomal expression was partially involvedin transgene expression observed in this study.

PGCs prior to their erasure of DNA methyla-tion have been used as a source of nuclear donorcells to successfully produce clone mice (Miki etal., 2005; Yamazaki et al., 2003). Genome-widedemethylation of DNA occurs during PGC mi-gration similar to the phenomenon during preim-plantation development of embryos. Hence, NTembryos using EG cells rather than somatic cellsmay be close to embryos from normal fertiliza-tion in their DNA methylation status, and thismay contribute the increased blastocyst develop-ment of NT embryos derived from EG cells. How-ever, further studies will be required to moreclosely demonstrate the effect of DNA methyla-tion of nuclear donor cells on the development ofNT embryos.

In conclusion, the present study demonstratedthat exogenous DNA can be efficiently intro-duced into porcine EG cells, and these cells canbe stably maintained in culture and used forkaryoplasts in NT. The porcine EG cell NT in-creased the efficiency of clone embryo productioncompared with conventional somatic cell NT.Whether this new procedure can be used to sup-port term development of NT embryos is re-mained to be determined by further studies.

ACKNOWLEDGMENTS

This work was supported by a grant(0301034410) from BioGreen 21 Program, RuralDevelopment Administration, a grant (105166)

from Technology Development Program forAgriculture and Forestry, Ministry of Agricultureand Forestry, and a grant (F104AD010004-06A0401-00411) from the KBRDG Initiative Re-search Program, Ministry of Science and Tech-nology, Republic of Korea.

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Address reprint requests to:Dr. Hosup Shim

Department of PhysiologyDankook University School of Medicine

San 29 Anseo-dongCheonan, Chungnam 330-714

Korea

E-mail: shim@dku.edu

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