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Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts¹

^a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211 ^b Department of Veterinary Medicine, College of Animal Resource Science, Kangwon National University, Chunchon 200-701, Korea ^c State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China ^d Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, Missouri 65211

	ABSTRACT

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Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

cloning, green fluorescent protein, pig, somatic cell, transgenic

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Applications for transgenic animal species range from tools in basic research to production agriculture. One major hurdle to improving this technology from its present state is to understand the mechanisms that affect transgene expression. Accurately predicting the transgene expression pattern in a genetically modified animal is vitally important in generating animal models for human disease as well as in designing animal bioreactors for harvesting secreted proteins.

Recent progress in animal cloning by somatic cell nuclear transfer (NT) has allowed researchers to produce animals from genetically defined backgrounds by replacing the genetic material of an unfertilized oocyte with that of a differentiated cell. This technique allows researchers to produce not only multiple genetically identical offspring, but also transgenic offspring by introducing foreign genes into the donor cells before NT. Specifically, progress has recently been made in the cloning of pigs by using somatic cell NT [1–3], and recently, genetically manipulated donor cells have been used as donors, which has resulted in transgenic offspring [4]; however, NT technology is still very inefficient.

Nuclear remodeling or reprogramming (or the lack thereof) has been described to some degree in nuclei transferred to enucleated oocytes [5, 6], but the mechanisms that are responsible for this are still not clearly defined. Although eGFP has previously been used successfully as marker of transgenesis and gene expression [4, 7–14], expression has been consistent. In 2000, two transgenic pigs were born that had been derived by injecting an eGFP gene into the perivitelline space of a meiotic metaphase II oocyte, which resulted in fertilization, and was followed by embryo transfer [15]. The ear skin fibroblasts from one of those transgenic pigs expressed eGFP. This animal had one copy of the eGFP inserted in to its genome. In the present study, we examined the expression and developmental ability of this eGFP-expressing donor cell line, as well as another eGFP-expressing cell line that was derived from a fetus at Day 35 after NT.

	MATERIALS AND METHODS

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Media

The medium used for oocyte maturation was tissue culture medium (TCM) 199 (31100035; Gibco, Grand Island, NY) supplemented with 0.1% polyvinyl alcohol (PVA), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 µg/ml LH (L-5269; Sigma Chemical Company, St. Louis, MO), 0.5 µg/ml FSH (F-2293; Sigma), 10 ng/ml epidermal growth factor (E-4127; Sigma), 75 µg/ml penicillin G, and 50 µg/ml streptomycin [16].

The medium used for enucleation was TCM 199 supplemented with 0.3% BSA (A-8022; Sigma) and 7.5 µg/ml cytochalasin B, and the medium for injection was the same medium, but without cytochalasin B. The medium used for activation consisted of 0.3 M mannitol, 1.0 mM CaCl₂·H₂O, 0.1 mM MgCl₂·6H₂O, and 0.5 mM Hepes.

The medium used to culture reconstructed embryos was North Carolina State University-23 medium [17] supplemented with 0.4% BSA.

Collection and Culture of Cumulus-Oocyte Complexes

Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% NaCl solution at 35–39°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (2–6 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe. COCs were washed three times in maturation medium, and 50–60 COCs were transferred to 500 µl of the same medium that had been covered with mineral oil in a 4-well multidish (Nunc, Roskilde, Denmark) and equilibrated at 39°C in an atmosphere of 5% CO₂ in air overnight.

Preparation of Ear Skin Fibroblasts and Fetal Fibroblasts

A small ear skin biopsy was obtained from the transgenic pig 402-2 [15] at 4 days of age, and the tissue was cut into small pieces with fine scissors. The cells were incubated for 30 min at 37°C in PBS containing 0.05% trypsin and 0.5 mM EDTA, and this suspension was centrifuged. The cell pellet was resuspended and cultured in Dulbecco modified Eagle medium supplemented with 75 µg/ml penicillin G, 50 µg/ml streptomycin, and 15% (v:v) fetal calf serum. The cells underwent this process up to four times. They were thawed, cultured, and then serum-starved (0.5% serum) for 3–5 days before NT (Fig. 1).

View larger version (120K): [in this window] [in a new window]   FIG. 1. Expression of eGFP in ear skin fibroblasts (EFs). A) EFs under normal light. A') EFs under an FITC filter set

The preparation of fetal fibroblasts and eGFP gene infection were conducted as previously reported [4, 14]. Fetal fibroblasts were obtained from a 35-day fetus and cultured in the same conditions that were used for the ear skin fibroblasts. To infect the cells, a replication-defective vector based on Moloney murine leukemia virus, pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) was used. Retroviral vector pseudotyped with VSV-G was carrying an eGFP gene under the control of the cytomegalovirus (CMV) promoter, LNCE-(VSV-G), which was kindly provided by Dr. A.W.S. Chan [11]. LNCE had long terminal repeat, neomycin-resistant gene, CMV promoter, and eGFP. Cells were infected with the retroviral vector by the following methods. Polybrene (0.1%) was diluted 1:30 with 0.1x HBS medium (22.9 mM Hepes, 140.3 mM NaCl, 0.7 mM NaH₂PO₄·H₂O). Three hundred microliters of the diluted medium were added to 4 µl of vector solution (10⁸ cfu/ml). The solution was diluted in 5 ml culture medium and incubated overnight. G-418 selection was started the following day, it continued for 13 days, and then the cells were frozen. The cells underwent this process up to seven times. The cells were thawed, cultured, and then serum-starved (0.5% serum) for 3–5 days before NT. This is the same cell line that was reported by Park et al. [4] that resulted in five cloned piglets and has resulted in a single cloned piglet when synchronized in G2/M before NT (unpublished).

Micromanipulation

After 42 to 44 h of culture, oocytes were freed from cumulus cells by vigorous vortexing for 4 min in TL-Hepes supplemented with 0.1% PVA and 0.1% hyaluronidase. Cumulus-free (denuded) oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm in enucleation medium with a glass pipette 30 µm in diameter. A single donor cell was placed in the perivitelline space of the oocyte to contact the oocyte membrane [4, 14].

Fusion/Activation of Oocytes

Injected oocytes were placed between two 0.2-mm diameter platinum electrodes 1 mm apart in activation medium. Fusion/activation was induced with two successive DC pulses of 1.2 kV/cm for 30 µsec on a BTX Elector-Cell Manipulator 200 (BTX, San Diego, CA). Nonmanipulated oocytes were electrically activated by using the same pulse parameters and cultured as controls.

Culture of Embryos

After fusion/activation, 20–30 reconstructed embryos were transferred to a 50-µl drop of culture medium covered with mineral oil in a 35-mm dish, and the dishes were held in 5% CO₂ in air at 39°C. Nonmanipulated oocytes were electrically activated and cultured as controls. Some reconstructed embryos were stained with 5 µg/ml bisbenzimide (Hoechst 33342) to identify nuclei by using an epifluorescent microscope (Nikon, Japan). After 6 days of culture all embryos were stained with Hoechst 33342 to determine the number of nuclei by using an epifluorescent microscope, and embryos with two or more nuclei were determined to have cleaved. To detect eGFP expression, embryos were examined on an epifluorescent microscope using a standard fluorescein isothiocyanate (FITC) filter set.

Embryo Transfer

To eliminate any potential of a culture-induced detrimental affect on development, embryos (1- to 2-cell stage) that had been cultured for 3–5 h or for 1 day (or both) after fusion were surgically transferred into one oviduct of each gilt. Pregnancy status was monitored by using an ultrasound scanner.

Polymerase Chain Reaction Analysis for the EGFP Gene

Genomic ear skin DNA from four NT piglets, a donor pig (402-2), and the surrogate mother was extracted and subjected to polymerase chain reaction (PCR) analysis using two sets of primers. For the eGFP gene, the eGFP forward primer (5'-CGCACCATCTTCTTCAAGGACGAC-3') and the reverse primer (5'-AACTCCAGCAGGACCATGTGATCG-3') were used. A 383-base pair (bp) amplicon was generated after eGFP gene amplification. PCR consisted of 32 cycles at 94°C for 45 sec, 61°C for 30 sec, and 72°C for 45 sec. PCR products were run on a 1% agarose gel [14].

Microsatellite Analysis

Genomic DNA from four NT piglets, a donor pig (402-2), and the surrogate mother was extracted and subjected to PCR. The microsatellite analysis consisted of three polymorphic porcine loci consisting of different multimers of dinucleotide repeats. The PCR profile included 5 min at 95°C followed by 35 cycles of 30 sec at 94°C, 45 sec at 62°C, and 90 sec at 72°C. PCR products were analyzed simultaneously on 6% polyacrylamide gels on an automatic sequencer using the Genescan and Genotyper software [14].

Experimental Design

The in vitro developmental ability of the NT embryos derived from ear skin fibroblasts and fetal fibroblasts was compared. After fusion/activation, the reconstructed embryos were cultured for 6 days, examined, and stained in order to count the number of nuclei. In addition, eGFP expression of embryos was examined. The in vivo developmental ability was also determined after transfer to surrogate gilts.

Statistical Analysis

Data were analyzed by ANOVA and the Duncan multiple range test by using general linear models in the Statistical Analysis System program to determine treatment differences. All percentage data were subjected to arcsine transformation before statistical analysis. Data are expressed as mean ± SEM. A probability of P < 0.05 was considered to be statistically significant.

	RESULTS

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In vitro developmental ability was determined after 6 days in vitro. The blastocyst formation percentage of control parthenogenetic embryos (26.1% ± 2.7%) was higher (P < 0.05) than either NT group (9.4% ± 1.5% and 10.0% ± 1.2%). The mean cell number of blastocysts (28.8 ± 2.8 to 33.5 ± 2.2) was not different (P > 0.05) among control and NT groups. In the NT-fetal fibroblast (NT-FF) group all embryos expressed fluorescence, but in the NT-ear skin fibroblast (NT-EF) group only 26.4% of cleaved embryos expressed green fluorescence. Among embryos expressing eGFP, 58.9% appeared mosaic in the NT-EF group (Table 1, Fig. 2).

View larger version (95K): [in this window] [in a new window]   FIG. 2. Expression of eGFP in NT embryos derived from ear skin fibroblasts. A–F) Embryos stained with Hoechst 33342 under UV light to identify the nuclei number. A'–F') Embryos under FITC filters to identify eGFP expression. A–B') Four-cell stage embryos. C–D') Eight-cell stage embryos. E–F') Blastocyst stage embryos. A', C', and E') Whole eGFP expression. B', D', and F') Mosaic eGFP expression

NT-EF embryos were surgically transferred to five surrogate gilts, and one gilt delivered four female piglets (NT7 to NT10) on Day 113 of gestation by caesarian method (Table 2). Birth weights ranged from 850 g to 1300 g, and placental weights ranged from 189 g to 374 g. Microsatellite analysis by using three markers suggested that all four piglets were derived from the ear skin fibroblast cell line (Table 3). PCR results confirmed the presence of the eGFP gene in all piglets and the donor cell line (Fig. 3). All four gilts expressed eGFP, as did the original donor [15]. All had coloration of the snout, oral and hoof wall of each limb as the former report. NT8 died at 3 days of age from a bacterial infection, and NT9 died at 7 days of age due to congestive heart failure.

View larger version (74K): [in this window] [in a new window]   FIG. 3. PCR analysis for eGFP gene in the surrogate and offspring

	DISCUSSION

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This study demonstrated that gene expression in all the blastomeres of an NT embryo is not uniform, and that NT technology by using ear skin fibroblasts can be used to duplicate transgenic pigs without transgene changes.

Ear skin fibroblasts from a transgenic pig [15] expressed eGFP (Fig. 1). After NT using these ear skin fibroblasts, eGFP was detected in 26% of the embryos from the two-cell stage to the blastocyst stage (Table 1). More interestingly, among eGFP-expressing embryos, more than half (59%) appeared mosaic; some blastomeres expressed eGFP, but some, which had nuclei, did not express well (Fig. 2). In contrast to the NT-EF group, mosaic expression was not observed in the NT-FF group (Table 1), nor when other groups used eGFP gene transduced somatic cell for porcine NT [12, 13]. To our knowledge, there are no reports of mosaic expression of eGFP or any other gene in NT embryos. The apparent mosaic expression in NT-EF was detected from the two-cell stage to the blastocyst stage.

Nuclear remodeling or reprogramming can be evaluated by studying the change in nuclear structure as well as the expression of specific genes [18]. Winger et al. [19] found that lactate dehydrogenase, citrate synthase, and phosphofructokinase were all correctly reprogrammed. In another study, it was shown by differential display technology that 95% of the transcripts in NT blastocysts were similar to the control, in vitro-produced embryo [20]. However, that also means that 5% of the transcripts were different. Apparently, Daniels et al. [21] have identified some of this 5%. They show that IL6, FGF4, and FGFr2 are not expressed correctly after NT. Thus, although much remodeling occurs normally after nuclear transfer, in some cases it is not complete.

Therefore, we examined the fluorescent expression of NT embryos at earlier time points. At 18 h postfusion 5% (n = 19) of NT embryos expressed fluorescence, and at 3 days postfusion, 8% (n = 51) of cleaved embryos expressed green fluorescence (unpublished data). But embryos with a mosaic expression were not detected at these stages. During culture, it is possible that some blastomeres may die and stop producing eGFP. Another possibility is that eGFP gene expression, as well as that of other genes, is independent in individual blastomeres. In this case, only some blastomeres would express eGFP. Genetic mosaicism is not likely because these embryos are derived from a single donor. Indeed, the animal from which the cells were derived has one integration site (Greg Bleck, personal communication) and all cells fluoresce (Fig. 1), which further suggests that she is not mosaic. Therefore, these results suggest that the gene expression pattern of NT embryos could be slightly different in each blastomere even though blastomeres were derived from a single cell and could be due to the site of integration. More work is needed to determine the exact control of gene expression. When the offspring of 402-2 is sufficiently expanded, then expression of this transgene during normal embryogenesis can be determined and directly compared with expression in in vitro-derived and NT-derived embryos.

Recently, a transgenic pig has been cloned from ear skin fibroblasts [22]. However, those researchers used in vivo matured oocytes for recipient cytoplasts, and in vitro culture data were not available. Therefore, in vitro developmental ability of NT embryos derived from ear skin fibroblasts was conducted by using in vitro matured oocytes. The developmental ability of NT embryos was not different between fetal fibroblast and ear skin fibroblasts as donor cells. However the percentage of blastocyst formation was still low (about 10%, Table 1).

Increased placental and embryonic weights have been reported in cloned calves, lambs, and mice [23–25]. However, the birth weights of these piglets (1300, 1300, 1300, and 850 g, respectively), if anything, were lower than normal for our recipient sow herd (1450 g). These weights are also similar to those reported by Park et al. [4]. We did not find any placental abnormalities. Unfortunately, two piglets died. NT8 had contracture of the flexor tendon of one front limb and died at 3 days of age due to a bacterial infection. NT9 died at 7 days of age from congestive heart failure. Her birth weight (850 g) was the lightest among the litter. NT7 had a hoof deformity; one of the dewclaws of the front left leg was abnormally large, and the front right leg had three dewclaws (polydactylia, Fig. 4). It is interesting that 402-2 had a contracture of the flexor tendons of her front legs at birth. She (402-2) was the result of in vitro embryo production and culture to the blastocyst stage before embryo transfer, and, to our knowledge, the first pig resulting from such a procedure. She has since given birth, and none of her offspring exhibit this phenotype.

View larger version (135K): [in this window] [in a new window]   FIG. 4. Two NT piglets (NT7 and NT10) derived from ear skin fibroblasts of the transgenic pig. NT7 had abnormal feet. One of the dewclaws of the front left leg was abnormally large (short arrow), and the front right leg had three dewclaws (long arrow shows additional dewclaw)

Low survival rate and deformities arising from the NT procedure is presumed to be a result of inappropriate reprogramming. In addition, highly abnormal methylation patterns in various genomic regions of cloned embryos was found and similar developmental abnormalities of cloned animals could be due to incomplete epigenetic reprogramming of donor DNA [5, 6, 26–28].

The number of nuclei in these NT-derived embryos appears to be low. In a recent paper of ours, an average nuclear number of 39 was reported from cells that resulted in term development [4], whereas here, the same cell line resulted in a nuclear number of 33. In addition, a nuclear number of 30 from embryos cultured in vitro can make fetuses [29].

In this study, we showed that expression of an eGFP transgene is not uniform between or within embryos, and that transgenic pigs can be duplicated by NT technology without a change in the transgene. The rates of term development and subsequent survival are still low. The efficiency can likely be improved by further studies into the epigenetic factors that affect embryo development.

	ACKNOWLEDGMENTS

 
We thank Max Rothschild, the U.S. Pig Genome Coordinator, for the primers for the microsatellites. We also thank Ed Brown and Daniel Liske for caring for the surrogate gilts during gestation; Tom Cantley for help with surgical embryo transfers; and Kristin Whitworth, Rami Woods, Jennifer Luth, Lisa Overman, and Dave Wax for helping care for the piglets after delivery.

	FOOTNOTES

 
First decision: 13 November 2001.

¹ We acknowledge funding from the F.B. Miller Fund (Department of Animal Sciences, University of Missouri-Columbia) to H.T.C., from the National Institutes of Health (NIH) via R01 RR13428 to R.S.P. and B.N.D., and from Food for the 21st Century. D.B.C. is the recipient of a Pathobiology Fellowship via T32 RR07004 funding from NIH. This work is contribution 13 184 in the Missouri Agricultural Experiment Station journal series.

² Correspondence: Randall Prather, University of Missouri, 162 ASRC, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827; pratherr{at}missouri.edu

Accepted: November 15, 2001.

Received: October 15, 2001.

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