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Chinese Science Bulletin Vol. 49 No. 2 January 2004 161

Chinese Science Bulletin 2004 Vol. 49 No. 2 161 166

Production of transgenic

calves by somatic cell

nuclear transfer

GONG Guochun1*, DAI Yunping

1*, FAN Baoliang1,

ZHU Huabing2, WANG Lili

1, WANG Haiping

3,

TANG Bo3, LIU Ying

3, LI Rong

3, WAN Rong

3,

HUANG Yinghua1 & LI Ning

1

1. State Key Laboratory for Agrobiotechnology, China Agricultural Uni-

versity, Beijing 100094, China;

2. Institute of Animal Science, Chinese Academy of Agricultural Science,

Beijing 100094, China;

3. Gentitan Biotechnology Ltd., Beijing 100084, China

* These authors contribute equally to this work.

Correspondence should be addressed to Li Ning (e-mail ninglbau@

public3.bat.net.cn)

Abstract Bovine fetal oviduct epithelial cells were trans-

fected with constructed double marker selective vector

(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced

green fluorescent protein (EGFP) and neomycin-resistant

(Neor) genes by electroporation, and a transgenic cell line

was obtained. Somatic cell nuclear transfer (SCNT) was car-

ried out using the transgenic cells as nuclei donor. A total of

424 SCNT embryos were reconstructed and 208 (49.1%) of

them developed to blastocyst stage. 17 blastocysts on D 7

after reconstruction were transferred to 17 surrogate calves,

and 5 (29.4%) recipients were found to be pregnant. Three of

them maintained to term and delivered three cloned calves.

PCR and Southern blot analysis confirmed the integration of

transgene in all of the three cloned calves. In addition,

expression of EGFP was detected in biopsy isolated from the

transgenic cloned calves and fibroblasts derived from the

biopsy. Our results suggest that transgenic calves could be

efficiently produced by SCNT using transgenic cells as nuclei

donor. Furthermore, all cloned animals could be ensured to

be transgenic by efficiently pre-screening transgenic cells and

SCNT embryos using the constructed double marker selec-

tive vector.

Keywords: transgenic, somatic cell, nuclear transfer, EGFP, bovine.

DOI: 10.1360/03wc0483

Transgenic large domestic animals have many poten-

tial applications in production of therapeutic and nutri-

tional protein, xenotransplantation and basic research.

Pronuclear DNA microinjection, the major method of

producing transgenic large livestock, was first applied to

production of transgenic mice[1] and later adapted to

transgenic livestock[2]. However, the low efficiency (1%

5%)[3], high cost and high percentage of chimera in off-

spring of this method have actually been the obstacles to

broaden its further application, especially to produce

transgenic large domestic animals. First, high cost is in-

volved in recovering enough embryos at pronuclear stage

either surgically or after slaughter of superovulated donors.

In addition, numerous surrogate females should be em-

ployed in production of one transgenic animal for the low

efficiency. Finally, long time of interval between the ob-

tainment of a founder animal and establishment of a lacta-

tion herd increases the cost of this method again. Many

studies toward animal transgenic have been mostly carried

out in mice and to a lesser extent in large livestock be-

cause of the lower efficiency[4] and higher cost of pronu-

clear microinjection in large domestic animals than in

mice.

Success of somatic cell nuclear transfer (SCNT) in

mammal[5] provides a more feasible approach to produce

transgenic livestock than the traditional pronuclear DNA

microinjection for its higher efficiency and lower cost.

Moreover, SCNT opens a way to homologous recombina-

tion and knock-in/knock-out technologies in large domes-

tic animals, which have been restricted to mice till now.

So far, transgenic sheep[6], cattle[7], goat[8] and gene tar-

geted sheep[9], pig[10 12] were produced successfully by

SCNT using transgenic somatic cells as nuclei donor. But

there were few reports[13,14] in production of transgenic

large livestock by SCNT in China. The aim of the present

study was to achieve high efficiency of transgenic calves

production by efficiently prescreening SCNT blastocysts

using EGFP in the constructed double marker selective

vector. To our knowledge, the present paper is the first

report about production of live transgenic cloned calves in

China.

1 Materials and methods

( ) Chemicals and materials. Consumables for cell

and embryo culture were purchased from Costar and Nunc

Company; unless stated otherwise, all chemicals for this

study were purchased from Sigma Company.

( ) Preparation of plasmid DNA. Construction of

pCE-EGFP-IRES-Neo-dNdB (Fig. 1), a double marker

selective vector, was described in detail by Gong et al.[15].

Plasmid was digested with Ssp and BamH to re-

move the backbone and then purified with QIAEXII kit

(Qiagen).

( ) Isolation of bovine fetal oviduct epithelial cells.

Tissue biopsies were obtained from the oviduct of a

D 147 fetus and washed twice with DPBS (Gibco). The

tissue biopsy was cut into small pieces, and tissue explants

were cultured in a 25 cm2 tissue culture flask containing 6

mL DMEM/F12 (Gibco) supplemented with 10% fetal

bovine serum (FBS; Hyclone) at 37 , 5%CO2 in a hu-

midified atmosphere. When the cells reached confluence

after 6 7 d, they were passaged twice and frozen in

DMEM/F12 supplemented with 20% FBS and 10% di-

methylsulfoxide (DMSO).

( ) Cell transfection and selection. After 2 3

passages, cells were suspended in Hepes-buffered saline

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Fig. 1. Schematic presentation of linearized double marker selective vector. CMV-IE enhancer, Enhancer of the encephalomyocarditis virus; pEF321,

the human elongation factor 1 promoter; EGFP, enhanced green fluorescent protein; IVS, synthetic intron; IRES, internal ribosome entry site; Neor,

neomycin resistance gene; poly A, SV40 early poly (A) signal.

(140 mmol/L NaCl, 5 mmol/L KCl, 0.75 mmol/L Na2HPO4,

6 mmol/L Glucose, 25 mmol/L Hepes) and the cell sus-

pension (0.8 mL) was mixed in an electroporation cuvette

(BTX) with 16 g of linearized pCE-EGFP-IRES-Neo-

dNdB. Cells were electroporated by a DC pulse of 1.2

kV/cm for 1 ms. 48 h after the electroporation, the cells

were incubated in medium containing 800 g/mL G418

(Gibco) for 14 d. The G418-resistant colonies were pooled

and cultured for further 4 5 passages. Before being used

as donor cells for NT, the cells were starved in culture

medium supplemented with 0.5% FBS for 2 4 d.

( ) In vitro maturation of oocytes. COCs with a

compact, nonatretic cumulus oophorus-corona radiata and

a homogeneous ooplasm were collected and washed twice

in maturation medium. Then 50 60 COCs were trans-

ferred into 0.5 mL maturation medium in 4-well dishes

overlaid with paraffin oil. The maturation medium com-

prised M199 (Gibco) supplemented with 10% FBS, 0.01

U/mL bFSH, 0.01 U/mL bLH, 1 g/mL estradiol and 1%

(v v) penicillin/streptomycin. COCs were cultured at

38.5 , 5% CO2 in a humidified atmosphere for 18 20 h.

After maturation, the cumulus cells were completely re-

moved by vortexing COCs in 0.1% hyaluronidase for 2

3 min.

( ) Nuclear transfer and in vitro culture of NT em-

bryos. Enucleation was performed under M199 contain-

ing 7.5 g/mL cytochalasin B and by using a microma-

nipulator equipped with an inverted microscope. Matu-

rated oocytes were enucleated with a 20 m (internal di-

ameter) micropipette by aspirating the first polar body and

partial surrounding cytoplasm presumably containing

metaphase chromosomes. Enucleated oocytes were

kept in M199 supplemented with 20% FBS until NT.

Immediately before NT, a suspension of the donor

cells was prepared by standard trypsinization. The cells

were pelleted and re-suspended in DPBS supplemented

with 0.5% FBS and remained in this medium until NT. A

20- m (in internal diameter) injection pipette containing a

donor cell was introduced through the slit of the zona pel-

lucida made during enucleation, and the cell was placed

between the zona pellucida and the cytoplast membrane to

facilitate close membrane contact for subsequent fusion.

Reconstructed embryos were electrically fused 24 h after

the start of maturation in buffer comprising 0.3 mmol/L

mannitol, 0.15 mmol/L CaCl2, 0.15 mmol/L MgCl2. Fu-

sion was performed in a chamber filled with fusion buffer

and with two stainless steel electrodes. The reconstructed

embryos were manually aligned with a fine glass needle

so that the contact surface between the cytoplast and the

donor cell was parallel to electrodes. Cell fusion was in-

duced with two DC pulses of 2.5 kV/cm for 10 s each,

1 s apart, delivered by a BTX Electrocell Manipulator

2001. After the electrical stimulus, the reconstructed em-

bryos were washed several times and incubated in M199

supplemented with 10% FBS for 30 min, and then exam-

ined for fusion by light microscopy.

The activation was induced by incubation in 5

mol/L ionomycin in CR1aa for 4 min at 37 . Embryos

were then extensively washed in CR1aa for 5 min before

culture in 1.9 mmol/L 6-dimethylaminopurine (6-DMAP)

in CR1aa for 4 h. Embryo culture was performed in 0.5

mL CRlaa supplemented with 5% FBS in 4-well dishes

overlaid with paraffin oil at 38.5 , 5% CO2 in a humidi-

fied atmosphere. On D 7.5 post fusion, the development of

embryos blastocyst stage was recorded.

( ) Embryo transfer. Partial of D 7 NT blastocysts

were transferred to synchronous recipients of 7 d after

standing estrus. The recipients were examined on D 30

after embryo transfer by palpation per rectum at D 60 and

90 of gestation for the presence of gestation.

( ) Detection of transgene in cloned calves by PCR

and Southern blot. Tissue fragments were obtained from

an ear punch of each newborn transgenic cloned calf, a

non-transgenic cloned calf and a Hostein cow selected

randomly. The genomic DNA samples were isolated from

these tissue fragments by phenol-chloroform extraction,

dissolved in Tris-EDTA buffer (TE) and used for PCR and

Southern blotting.

(1) PCR. GFP-specific primers (forword: 5 - TGC-

AGTGCTTCAGCCGCTAC-3 ; reverse: 5 -CTCAGGT-

AGTGGTTGTCGGG-3 ) were used to amplify a 384-

base fragment in the GFP-reporter gene. The PCR reac-

tions were run for 35 cycles, with denaturation at 95

for 30 s, annealing at 62 for 30 s, followed by exten-

sion at 72 for 30 s. A final extension for 10 min at 72

was included after the last cycle. Following amplification,

162 Chinese Science Bulletin Vol. 49 No. 2 January 2004

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the products were analyzed on a 1.5% agarose gel stained

with ethidium bromide.

(2) Southern blotting analysis. It was carried out by

standard procedures. Purified genomic DNA (10 g) was

digested with Bgl and separated on a 0.7% agarose gel

in tris-acetate buffer, transferred onto a nylon membrane,

cross-linked by ultraviolet light, then hybridized with a

probe of 1.6 kb, which was Bgl -Bgl DNA fragment

of pCE-EGFP-IRES-Neo-dNdB labeled with -32P-

deoxycytidine triphosphate (dCTP).

( ) Microsatellite analysis. Microsatellite analysis

was performed on the calves produced by SCNT and the

surrogate recipient females to confirm genetic identity

with the donor cells used for SCNT. Genomic DNA was

extracted from the transfected and non-transfected donor

cells, tissue fragments of the transgenic cloned calves, the

surrogate recipient females and a Chinese yellow calf se-

lected randomly. Microsatellite assay was carried out us-

ing 22 bovine DNA microsatellite markers (INRA063,

HEL9, HEL1, INRA035, ETH225, ILSTS005, INRA023,

BM2113, BM1824, INRA0325, MM12, HAUT24,

HEL135, ILSTS006, HEL51, INRA037, INRA005,

ETH104, TGLA227, TGLA122, ETH152 and ETH3).

Length variations were assayed by polymerase chain reac-

tion (PCR) amplification with fluorescently labeled lo-

cus-specific primers and PAGE on an automated DNA

sequencer (ABI 373: Applied Biosystems, Foster city,

CA). Proprietary software (GeneScan and Genotyper; Ap-

plied Biosystems) was used to estimate PCR product size

in nucleotide.

( ) Expressing of GFP in NT embryos and trans-

genic cloned calf. Part of the reconstructed embryos

derived from transgenic fetal oviduct epithelial cells were

checked by brief exposition to blue light to determine if

GFP was expressed when developed in to different stages.

A skin biopsy was surgically obtained from the ear of

the living transgenic cloned calf. Partial of the biopsy was

cut into very small pieces. A drop of PBS containing the

biopsy pieces was placed in the centre of a slide and a

cover slip was placed over the drop. Expression of GFP in

the skin biopsy was detected under blue light. The other

partial of the biopsy was immersed in 70% ethanol for 1

min, rinsed twice with Ca2+- and Mg2+-free PBS, cut into

small pieces and paved in a 25-cm2 tissue culture flask

containing 6 mL Dulbecco modified Eagle medium

(DMEM; Life Technolngies) supplemented with 10% (v/v)

FBS. Fibroblasts derived from the biopsy were examined

to observe the expression of GFP under blue light.

2 Results

( ) Expression of GFP in SCNT embryos. Ex-

pression of GFP was detected at the late 8 16-cell stage

in the majority of SCNT embryos and at 2-cell stage in the

minority of SCNT embryos. Furthermore, the expression

intensity of GFP increased gradually with the development

of SCNT embryos and reached the peak at blastocyst stage

(Fig. 2).

( ) Development of transgenic cloned embryos. A

total of 424 SCNT embryos were reconstructed and 208

(49.1%) of them developed into blastocyst stage. 17 blas-

tocysts on D 7 after reconstruction were transferred to 17

surrogate calves and 5 (29.4%) recipients were found to

Fig. 2. Expression of GFP in transgenic SCNT embryos cultured in vitro. (a) and (b) NT embryos at 8 16-cell stage; (c) and (d) at morula stage; (e)

and (f) blastocysts on D 7 after fusion. (a), (c) and (e) are images under conventional light whereas (b), (d) and (f) under blue light.

Chinese Science Bulletin Vol. 49 No. 2 January 2004 163

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Fig. 3. PCR assay of transgenic cloned calves. M, 1 kb ladder; 1 3,

transgenic cloned calves; 4, non-transgenic cloned calf; 5, Holstein cow; 6,

plasmid pCE-EGFP-IRES-Neo-dNdB; 7, distilled water.

be pregnant. Three of them maintained to term and deliv-

ered three cloned calves (15%).

( ) Identification of the transgene by PCR and

Southern blot. Analysis of genomic DNA by PCR (Fig.

3) and Southern blot assay (Fig. 4) confirmed the integra-

tion of transgene in all of the three cloned calves. The

possibility of cross-contamination was excluded because

no products were detected in the negative control in PCR

and Southern blot assay.

( ) Microsatellite analysis of transgenic cloned

calves. Microsatellite DNA analysis of the examination

of 22 loci confirmed that the cloned calves were geneti-

Fig. 4. Southern blot assay of transgenic cloned calves. M, 1 kb ladder;

1 3, transgenic cloned calves; 4, non-transgenic cloned calf; 5, Holstein

cow; 6 8, plasmid pCE-EGFP-IRES-Neo-dNdB (1 copy, 5 copies and 10

copied respectively).

cally identical to the donor cows and different from the

recipient females and Chinese yellow calf selected ran-

domly (Table 1).

( ) Expression of GFP in skin biopsy and fibro-

blasts derived from transgenic cloned calves. Expression

of GFP was observed in skin biopsy isolated from the ears

of transgenic cloned calves (Fig. 5(a), (b)).

Fibroblast cell line was established from biopsy iso-

lated from the ears of transgenic cloned calves. Expression

of GFP was detected in the minority but not detected in

the majority of these fibroblasts (Fig. 5(c), (d)).

Table 1 Microsatellite analysis of transgenic cloned calvesa)

Marker

number

Marker

name 1 2 3 4 5 6 7 8 9

1 INRA063 177 177 177 177 177 177 177 177 177 177 175 177 177 177 171 173 165 167

2 HEL9 153 155 153 155 153 155 153 155 153 155 149 153 161 169 157 165 151 159

3 HEL1 105 113 105 113 105 113 105 113 105 113 111 111 105 111 107 109 97 101

4 INRA035 102 102 102 102 102 102 102 102 102 102 104 104 102 104 100 118 102 112

5 ETH225 151 151 151 151 151 151 151 151 151 151 145 145 149 149 137 147 143 145

6 ILSTS005 184 184 184 184 184 184 184 184 184 184 184 186 184 186 178 182 180 182

7 INRA023 213 217 213 217 213 217 213 217 213 217 199 217 209 209 207 213 205 215

8 BM2113 138 150 138 150 138 150 138 150 138 150 136 152 136 150 122 132 126 130

9 BM1824 183 183 183 183 183 183 183 183 183 183 181 183 181 183 177 185 175 179

10 INRA0325 182 186 182 186 182 186 182 186 182 186 180 180 182 182 174 178 176 178

11 MM12 119 119 119 119 119 119 119 119 119 119 107 107 117 125 103 113 115 121

12 HAUT24 131 131 131 131 131 131 131 131 131 131 119 119 119 131 115 127 111 115

13 HEL135 190 194 190 194 190 194 190 194 190 194 194 194 186 190 188 188 188 192

14 ILSTS006 329 335 329 335 329 335 329 335 329 335 327 329 329 333 287 291 285 293

15 HEL51 163 167 163 167 163 167 163 167 163 167 153 155 163 167 165 169 155 169

16 INRA037 120 132 120 132 120 132 120 132 120 132 120 130 120 132 124 128 116 122

17 INRA0052 139 141 139 141 139 141 139 141 139 141 141 141 141 143 135 141 143 149

18 ETH104 213 217 213 217 213 217 213 217 213 217 219 219 219 225 211 223 215 221

19 TGLA227 83 99 83 99 83 99 83 99 83 99 95 95 85 105 81 93 89 91

20 TGLA122 152 152 152 152 152 152 152 152 152 152 152 152 152 152 154 154 146 156

21 ETH152 201 211 201 211 201 211 201 211 201 211 193 193 197 199 195 201 193 197

22 ETH3 124 126 124 126 124 126 124 126 124 126 116 124 116 116 114 122 118 126

a) 1 9 represent non-transgenic donor cell, transgenic donor cell, 3 cloned calves, Chinese yellow calf and 3 surrogate recipients respectively.

164 Chinese Science Bulletin Vol. 49 No. 2 January 2004

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Fig. 5. Expression of GFP in skin biopsy ((a) and (b)) from transgenic cloned calf and fibroblasts ((c) and (d)) derived from the biopsy. (a) and (b) are

images under conventional light whereas (c) and (d) under blue light.

3 Discussions

SCNT has many potential advantages when used to

produce transgenic large domestic animals contrasted with

other methods. Nonetheless, low efficiency of SCNT is

the major limitation of this approach. In addition to em-

bryo loss, SCNT is also associated with high rates of fetal

and neonatal loss, and production of abnormal offspring[16].

In the present study, high rate of fetal loss (40%) was also

one of the major factors that affected the efficiency of

SCNT. Report of Hill[17] indicated that abnormal devel-

opment of the placenta, including vascular reduction, is a

principal contributor to loss particularly during early

pregnancy in sheep and cattle. The reasons for the high

abortion rate are not clear but might be due to insufficient

reprogramming of the donor nuclei[18].

The advantage of using SCNT to produce transgenic

animals is the ability to use preselected genetically modi-

fied cells as donor nuclei. All of the animals created via

NT from such selected cells should be transgenic. How-

ever, results of recent studies[10,19,20] suggested that selec-

tion of transgenic cells using G418 was not sufficient to

completely remove nontransgenic cells in culture, and

cloned animals produced from these cells were not always

transgenic. In our study, transgenes were detected in all of

the three cloned calves derived from SCNT embryos pre-

screened by the constructed double marker selective vec-

tor. This result reveals that all cloned animals could be

transgenic when the constructed double marker selective

vector is employed in transgenic animal production by

SCNT. Furthermore, studies on the production of trans-

genic animals carrying other functional transgenes by the

constructed double marker selective vector are in pro-

gress.

Most of SCNT embryos were observed to express

GFP at the late 8 16-cell stage, which corresponds with

the period of major embryonic genome activation in bo-

vine embryos[21]. In addition, increase of GFP expression

level in SCNT embryos may be due to the accumulation of

green fluorescent protein with the development of these

embryos.

Observation in skin biopsy of transgenic calves and

fibroblasts derived from the biopsy indicated that GFP

was not expressed in all of the tissues or cells. It is possi-

ble that expression of the transgene is affected by epige-

netic modification during development and culture in vitro.

In similar study, Keefer et al.[22] did not observe the

expression of the GFP protein in both epithelial cells taken

from a mouth swab and skin fibroblasts of GFP transgenic

cloned goat. However, expression of GFP was induced in

partial of a subpopulation of the skin fibroblast cells after

Chinese Science Bulletin Vol. 49 No. 2 January 2004 165

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166 Chinese Science Bulletin Vol. 49 No. 2 January 2004

drug treatment with 5-azacytidine, a cytidine analogue

that inhibits methylation when incorporated into DNA.

This result indicated the presence of a functional trans-

gene that may have been silenced by epigenetic mecha-

nisms.

Acknowledgements We would like to thank the members in Shandong

Liangshan Animal Science Ltd. and Hebei Lutai Cattle Farm for their

preparation of the recipients, assistance in embryo transfer, care of the

newborn NT calves. The authors also thank Dr. Zheng Xiufeng, Xu

Gongjing for their contributions to microsatellite analysis, Zhang Lei for

NT calves delivery and husbandry, Zhang Yunhai and Pan Dengke for

helpful comments on the manuscript. This work was supported by the

State “863” High-Tech Research and Development Project (Grant Nos.

2002AA206111 and 2001AA213091), and the Natural Science Founda-

tion of Beijing (Grant No. 5030001).

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(Received September 25, 2003; accepted November 24, 2003)