Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.


Introduction
There are many ways available to produce transgenic animals, such as pronuclear injection and sperm-mediated gene transfer [1]. Somatic cell nuclear transfer (SCNT), which uses preselected genetically modified cells as donor nuclei, is recognized as a more efficient way to produce transgenic animals until now [2]. SCNT is widely used to generate transgenic pigs and cows which have improved meat or milk quality [3,4]. However, researches about the application of transgenic cloned goats are less. Dairy goats are preferable to other species to produce recombinant proteins in milk for the following reasons [5]. First, dairy goats produce more milk than rabbits and mice. Second, unlike cows, goats have earlier sexual maturation, shorter breeding generation interval, and more offspring per parity. Third, goat milk is safer for human, because there is no severe infectious disease like mad cow disease. Fourth, goats are more amendable to be cloned by NT and the cloned offspring have a longer life span. Human Lactoferrin (hLF) plays important physical roles in anti-bacterial, anti-viral, antifungal infections, and cancer prevention [6]. Many previous attempts have been intended to produce hLF for structure function studies [7,8], but the previous methods were not suitable for very large-scale production. Transgenic goat mammary gland is an alternative way of producing this protein, because of large milk yield and precise posttranslational modifications [9]. Thus, we decided to investigate the effective way of producing hLF transgenic cloned dairy goats.
Although SCNT is the preferred approach to produce transgenic goats and other large animals, the success percentage remains low [10]. Early embryonic and fetal loss, stillbirth, postnatal loss, and abnormalities account for this. The reason of low birth survival is probably due to incomplete reprogramming of epigenetic marks in differentiated donor cells [11]. DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming. Imprinted genes are epigenetically marked with their parental origin and a given parental allele is expressed while the other is repressed [12]. Knockdown or deletion experiments have demonstrated that imprinted genes participate in ongoing development, such as energy metabolism in life cycles [13]. However, the expression level of imprinted genes was mainly affected by the DNA methylation [14]. Aberrant imprinting has been shown to result in a number of diseases, including a variety of developmental syndromes, such as Prader-Willi syndrome and cancers [15]. During embryonic development, the methylation status of some imprinted genes is dynamic and has spatial and temporal requirements [12]. H19 and Insulin-like growth factor 2 receptor (IGF2R) are essential for normal development and therefore the most studied imprinted genes involved in foetal growth regulation. Abnormal DNA methylation of differential methylated regions (DMR) in imprinted genes may cause their biallelic expression or silencing [12].
Many details of DNA methylation of imprinted genes have been elucidated in cloned mice [16] and swine [12,17]. However, little is known in ruminants, especially for cloned goats, due to lack of genomic sequence [18]. Goat is an economically important agricultural species and animal model for biology studies as well. The interest in elucidating imprinting mechanisms in ruminants has grown in recent years due to the great incidence of abnormal development in animals produced from assisted reproductive technologies such as SCNT. Additionally, along with the more available whole-genome sequences from Goat Genome Project, it is possible to systematically study the event of methylation imprints in goats [18]. A more thorough understanding on how methylation imprints is reprogrammed during cloning and how the methylation is maintained during development will help to improve the technique of NT.
In this study, we produced five transgenic cloned goats successfully using SCNT. Three were born alive and the other 2 were dead during gestation. Furthermore, our study on the DNA methylation in live and dead transgenic cloned goats demonstrated that H19 and IGF2R played significant roles in the nuclei of donor cells reprogramming in NT, which could explain the death of transgenic cloned goats aborted in this study.

Construction of the hLF Mammary Gland-specific Expression Vector
The hLF cDNA (GenBank accession no. X53961) was artificially synthesized and inserted into the vector pBluescript II SK (+) by Invitrogen Service, (Shanghai, China). The vector also contained a signal peptide sequence, a coding area, a stop codon (TAA), and two XhoI restriction sites. The hLF cDNA sequence was isolated from pBluescript II SK (+) and inserted into the vector PBC1 (Invitrogen, USA) by XhoI site. The obtained vector was termed pBC1-hLF. 794 bp coding region of neomycin (neo) gene was obtained from pcDNA3.1 vector (Invitrogen, USA) and cloned into pIRES2-EGFP (Clontech, USA). The obtained vector was named pNeo-IRES2-EGFP. After deleting Sal I and Xho I sites, this vector was used as the template to amplify the fragment of Neo-IRES2-EGFP by PCR, PhusionH High-Fidelity DNA Polymerase (NEB, UK) and the following procedure: pre-denaturing at  98uC for 2 min, and 30 cycles with denaturing for 20 s at 98uC,  annealing for 30 s at 61uC, extension for 2 min at 72uC, and a  final extension for 8 min at 72uC. After sequencing, this fragment was cloned into the pBC1-hLF vector under the control of the cytomegalovirus promoter. The final vector was named as pBC1-hLF-Neo-IRES2-EGFP. The schematic diagram of this vector was shown in Figure 1. Primers used for the plasmid construction was listed in Table S1 in File S1.

Inductive Expression of hLF Mammary Gland-specific Expression Vector in vitro
We isolated the primary mammary epithelial cells (MECs) from a lactating dairy goat 60 days post parturition using the methods described in the previous study [19]. Anti-cytokeratin 18 (Abcam, USA), which is the epithelial cells specific protein, was used to identify this cell by immunofluorescence. The purified passage 4 goat MECs were transfected with the vector pBC1-hLF-Neo-IRES2-EGFP. The G418 resistant MECs were confluent in 24well plates through 4 weeks of G418 selection. Then cells were trypsinized and propagated for two passages under G418 selection. Passage 6 G418 resistant cells were obtained and used for inductive expression. When they were 80% confluent, the cell cultural medium was replaced by inductive medium (DMEM/ F12+10 ng/mL EGF +1% ITS +5 mg/mL prolactin +1 mg/mL hydrocortisone). Total RNA was isolated using RNA easy kit (Qiagen, USA), reverse-transcribed (Invitrogen, USA) and PCR amplified hLF with the GAPDH mRNA as a reference gene. Nontransgenic MECs were used as a negative control. The supernatants after induction were concentrated and subjected to Western blot by the standard protocol. The rabbit anti-human Lactoferrin antibody (1:1000, Abcam, USA) and the secondary antibody was donkey anti-rabbit IgG (1:5000, LI-COR, Gemany).

Preparation of Goat Fibroblasts
Goat fibroblasts cells (GFCs) were obtained from the ear of a 2month-old healthy female Saanen dairy goat (Yangling, China). The ear tissue was cut into small pieces. Tissue explants were cultured in dulbecco modified eagle medium (DMEM F-12, Sigma, USA), supplemented with 10% fetal bovine serum (FBS, Gbico, USA) and 1% (v:v) penicillin/streptomycin (10000 U/mL penicillin G, 10000 mg/mL streptomycin, Sigma, USA) at 37.8uC in 35-mm tissue culture plates in a humidified atmosphere of 5% CO 2 in air. Cultured for 10 days, the explants were removed, and cells were harvested by trypsinization, counted, and seeded in 75 cm 2 tissue culture flasks. When the cells reached confluency, they were collected by trypsinization and frozen in DMEM-F12 supplemented with 40% FBS and 10% dimethyl sulfoxide (DMSO, Sigma, USA).
The GFCs was identified using immunofluorescence. The cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and unspecific binding was blocked with a 2% solution of bovine serum albumin (BSA) in PBS for 1 h. The cells were then incubated with anti-Vimentin (Santa Cruz, USA) overnight at 4uC. Alexa Fluor 488-conjugated antibody was raised in goat and directed against immunoglobulins of the species used to raise the primary antibody, were used for detection of the bound primary antibodies (1:200, 1 h). Between each step the cells were rinsed with PBS. For a negative control, the entire procedure was followed except that the primary antibody was replaced with PBS. A light-scanning microscope was used to examine the cells.

Transfection, G418 and EGFP Selection of GFCs
Passage 4 fibroblast cells were transfected with the purified linearized vector pBC1-hLF-Neo-IRES2-EGFP with pvuI enzyme using the Lipofectamine LTX TM (Invitrogen, USA), following the manufacturer's guidelines. Briefly, 2610 5 cells at passage 4 were seeded in a 35-mm culture plate one day before transfection. Cells were transfected with 4 mg of DNA. After transfection, the cells were exposed to 1000 mg/ml of G418 (Gibco, USA) for 15 days. After that, 500 mg/ml of G418 was used during the selection process to obtain stable expression, and single colonies were isolated in the presence of G418 and expanded. Single transfected cell colonies were picked up by using cloning discs (Fisher Scientific, Springfield, Nanjing, China) and placed in 24-well plates. Cells in each well were examined for EGFP expression under fluorescent light using a standard fluorescein isothiocyanate (FITC) filter setting and marked as positive and negative cell lines. When confluence was achieved, cells were passaged to 6-well plates and then to 35-mm plates. A portion of the cells from each EGFP-positive cell lines were plated for DNA isolation, quantity of EGFP expression, and cell cycle distribution. The remaining cells were frozen in DMEM-F12 supplemented with 40% FBS and 10% (v:v) DMSO.

Identification of Transfected Cells by PCR and Cell Cycle Distribution
The G418-resistant and EGFP positive cell clones were analyzed by PCR to confirm transgene integration. Genomic DNA was isolated from each cell line using a Genomic DNA kit (Tiangen, China). PCR was used to identify the presence of hLF gene following procedure of pre-denaturing at 94uC for 5 min, and 30 cycles with denaturing for 30 s at 94uC, annealing for 30 s at 62uC, extension for 45 s at 72uC, and a final extension for 5 min at 72uC. To avoid non-specific amplification, the PCR primers were designed to amplify the 750 bp product covering part (1-342 bp) of the vector sequence and part (343-750 bp) of the hLF gene. The forward and reverse primer was 59-GAATGGCTGGCAGTGAAACA-39 and 59-CTCAATGGGCTCAGGTGGAC-39, respectively.
Cell cycle and EGFP expression analysis of transfected cells after PCR were analyzed by flow cytometry (FACS-CALIBUR, Becton Dickinson, Sunnyvale, CA). The cells were cultured in 6well plates and grown in DMEM containing 10% FBS until 100% confluent. Then the cells were cultured for additional 2 days into contact inhibition. They were harvested using 0.25% trypsine-EDTA (Sigma, China) and re-suspended in DMEM at 1610 7 cells/tube. The cells were re-suspended in PBS and centrifuged at 1,500 rpm at 4uC for 5 min. The supernatant was decanted, and the cells were gently re-suspended in PBS. Cells were fixed by adding 0.7 mL cold ethanol (70%) drop-wise in a tube containing 0.3 mL of cell suspension in PBS while gently vortexing. Fixed cells were left undisturbed at 4uC for 48 h before further analysis. The fixed cells were centrifuged at 1,500 rpm at 4uC for 5 min, washed once with cold PBS, and re-centrifuged. For expression analysis of the EGFP transgene, centrifuged cells were resuspended in 0.25 mL PBS and analyzed by flow cytometry with the instrument set for fluorescein detection. For cell cycle analysis, centrifuged cells were re-suspended in 0.25 mL PBS containing 5 mL of 10 mg/mL RNase (Sigma, USA) and incubated at 37uC for 1 h. Incubated cells were stained by adding 10 mL of 1 mg/mL propidiumiodide (Sigma, USA). The cells were then analyzed by FACS-CALIBUR at 488 nm. Non-transfected cells were used as a negative control for fluorescein and cell cycle detection. Histogram and dot plots were created using Cell Quest software (Beckton Dickinson, San Jose, CA). The percentages of cells cycle within the various phases of the cell cycle were calculated using the modfit program.

Nuclear and Embryo Transfer
Transfected cells, which were positive for PCR amplification for hLF, high EGFP stable expression, normal cell cycle distribution, were used as donor cells. Oocyte collection and enucleation, nuclear transfer, oocyte activation, embryo culture, and embryo transfer were carried out with protocols as described previously [15]. First, goat ovaries were collected from an abattoir (Er'Ling Goat Abattoir, Danyang, China) with the permission of using these ovaries for normal scientific research. Cumulus-oocyte complexes (COCs) were sorted under stereomicroscopy, and cultured for 20 to 23 h at 38.5uC under 5% CO 2 in humidified air. Matured oocytes were then pipetted with 0.3% hyaluronidase to remove granulosa cells. Those that had released their polar bodies were selected. Second, oocytes (15-20/group) were treated for 10 min in TCM199 with 7.5 g/mL cytochalasin B, 10 g/mL Hoechst 33342, and 10% FBS, and examined under fluorescence microscopy. Each oocyte was aspirated using a holding pipette. Only completely enucleated oocytes were used for NT. Third, a micropipette containing the donor cell was introduced through a slit made on the zona pellucida during enucleation. The cell was inserted between the zona and cytoplast membrane to facilitate close membrane contact and subsequent fusion. Reconstructed embryos were washed with fusion medium (0.25 M d-sorbitol, 0.5 mM (CH 3 COO) 2 MgN4H 2 O, 0.1 mM (CH 3 COO) 2 Ca, 0.5 mM HEPES, and 1 mg/mL BSA), and then moved to microslides (BTX, Inc., San Diego, CA, USA) with fusion medium. Fusion was induced with two 1.2 kV/cm DC pulses lasting for 20 s. The fusion results were examined after 0.5 h. The embryos that were not fused were fused again. Finally, reconstructed embryos were treated with TCM199 containing 5 mM ionomycin for 5 min, incubated for 4 h in TCM 199 containing 2 mM 6-DMAP, washed twice, transferred to SOFaa, and cultured in CO 2 incubator under 38.5uC, 5% CO 2 , and

Microsatellite Analysis, PCR and Southern Blot Analysis of Cloned Goats
Tissue collection and genomic DNA extraction. Ear tissues were obtained from new born goats from the present study, including 3 cloned lived goats (CL1, CL2, and CL3) and 2 cloned aborted goats (CA1 and CA2, aborted at 140 days of gestation). The ear tissue of an adult recipient goat from natural reproduction was collected as a control. All samples were frozen immediately after collection and stored at 280uC until further analysis. The Institutional Animal Care and Use Committee at Nanjing Agricultural University approved all procedures involving the use of animals. Genomic DNA was extracted from the ear tissues using a Genomic DNA Kit (Tiangen, Beijing, China) for the following experiments.
Microsatellite analysis. The five cloned goats obtained by SCNT were subjected to parentage analysis to confirm genetic identity with the donor cells used for SCNT. DNA was extracted from the ear tissues from the goats, surrogate recipients, and donor cells. Microsatellite assay used eight caprine and bovine microsatellite markers (ETH10, ETH152, ILSTS005, ILSTS008, INRA063, INRA011, CP34, MAF65) taged with fluorescent dyes FAM [20]. All of the PCR products were run on a 3100 Genetic Analyzer. The allele sizes were determined using Genescan software (ABI, USA). Primers for the eight Microsatellite markers were listed in Table S2 in File S1.

Genomic DNA Analyses with PCR and Southern Blot
To amplify the hLF gene by PCR, the nucleotide sequence of the pBC1 cloning vector (Invitrogen, USA) was used to design Identification primer I (Table S3 in File S1) corresponding to the pBC1 nucleotide sequence at positions 8,554-8,580 bp and 10,789-10,814 bp, respectively. This could yield a predicted amplification product of 2,237 bp. Each PCR reaction mixture contained 100 ng genomic DNA, 10 pmol of each primer, and 10 mL of 26G 0 Taq Green Master Mix (Promega, Madison, Germany). The PCR procedure was pre-denaturing at 95uC for 5 min, and 30 cycles with denaturing for 30 s at 95uC, annealing for 30 s at 58.8uC, extension for 30 s at 72uC, and a final extension for 7 min at 72uC.
For Southern blot analysis, genomic DNA (20 mg) was digested with Hind III. The fragments containing the full sequence of the hLF gene were separated on the 0.7% agarose gel. For blot analysis, primers for the synthesis of probe (Table S3 in File S1) were corresponding sequence positions 8,269-8,288 bp and 9,088-9,107 bp of the vector, respectively. The probe was synthesized using a PCR DIG Probe Synthesis kit (Roche, Germany), purified by agarose gel electrophoresis, and labeled with digoxigenin before hybridization. Detection of labeled DNA on the positively charged nylon membrane was performed using a DIG luminescent detection kit (Roche, Germany). Identification of putative DMRs of imprinted genes in goat genome. The DMRs of imprinted genes have regions that are highly conserved across various mammal species. Therefore, we compared the well-defined DMRs of H19 and IGF2R in human, bovine, and ovine genomes to identify the counterparts of these DMRs in goat genome, using an online software MethyPrimer with restrictive conditions (GC percentage .50.0%, CpG observe/expect .0.6) to identify the distribution of CpG islands.

The Methylation Status Analysis of Genomic DNA of Transgenic Cloned Goats
Bisulfite treatment of oligonucleotides and primers design. Bisulfite treatment and recovery of DNA samples were carried out with the EpiTect Bisulfite kit (Qiagen, USA), following the manufacturer's instructions. Briefly, 2 mg DNA in 20 mL volume was used for each reaction and mixed with 85 mL bisulfite mix and 35 mL DNA protect buffer. Bisulfite conversion was performed on a thermocycler as follows: 99uC for 5 min, 60uC for 25 min, 99uC for 5 min, 60uC for 85 min, 99uC for 5 min, 60uC for 175 min and 20uC indefinitely. The bisulfite-treated DNA was recovered by EpiTect spin column and subsequently sequenced to confirm the efficiency of bisulfite conversion.
The specific primers for amplification and sequencing of DMRs of H19 and IGF2R were designed using the Primer Express software application (ABI, CA). Primers for bisulfite treated DNA were optimized to amplify a fragments of 363 bp for H19 and a 244 bp for IGF2R at annealing temperature of 55-65uC. We designed the primer spanning the DMR encompassing CTCF3 site of H19 gene (F: 59-TATTAGGTTTTTGGTGGTATAGAG-39, R: 59-CTCTCCTCTCCCAACTTCAA C-39). For IGF2R, we designed the primers spanning the DMR in intron 2 (F: 59-GTTTTATGGTYGTYGGTAGAGGTA-39, R : 5 9-CRCCRAATCCTACAA ACCCTAC-39; Y = C/T, R = A/G).
Bisulfite PCR and sequencing. Touch Down PCR for H19 was performed with a 26PCR master mix solution (TaKaRa, JA) as following procedures: one cycle of 95uC for 5 min; two cycles of 95uC for 30 s/60uC (annealing Tm) for 1 min/72uC for 1 min; two cycles of 95uC for 30 s/58uC for 1 min/72uC for 1 min and so forth; lastly, 25 cycles of 95uC for 30 s/50uC (annealing Tm) for 1 min/72uC for 1 min, 72uC for 7 min. The PCR procedure for IGF2R was the same as H19, but the annealing Tm ranged from 62uC to 54uC. The PCR products were recovered and purified with Wizard SV Gel and PCR Clean-Up System (Promega, USA) following the manufacturer's protocol and subcloned into pMD19-T cloning vector (TaKaRa, JA). Positive transformants colonies (n = 15) were sequenced by Big Dye (ABI, USA) on an ABI PRISM Model 3730 and analyzed using the Sequencer software application version 4.1.4 (Gene Codes, USA). After bisulfite treatment, unmethylated cytosine residues turns into thymine residues, while methylated cytosine residues remain as cytosine residues. The methylation patterns were analyzed only using sequences derived from more than 10 clones with .99% cytosine conversions only, with BIQ Analyzer software. The methylation percentage of every sample was obtained from BIQ Analyzer software. DNA methylation among the three groups were tested by one-way ANOVA, statistical analyses were performed using GraphPad Prism (version 5.03, San Diego, USA). Differences were considered significant at P,0.05.

Selection of Competent Donor Cells for SCNT
Culture and identification of stable GFC lines. The GFCs were isolated and purified to be used as donor cells preparation ( Figure S1). After transfection with constructed vector and G418 selection, 64 G418-resistant cell clones were obtained. Then, 34 clones expressing EGFP (Figure 2A) were selected for PCR identification and resulted in 28 positive clones ( Figure 2B).
Cell cycle distribution after contact inhibition. Among 28 EGFP and PCR positive GFC clones, 23 were with good status and selected for the following cell cycle test. The results indicated that the average G0+G1 ratios of 19 cell clones were 83.45% (SE = 2.96%), which was not significantly different from the nontransfected control cells (87.26%, SE = 0.42%) ( Table S3 in File S1). These results confirmed that these 19 transgenic cell clones were competent as donor cells for SCNT.

Inductive Expression of hLF cDNA in vitro
hLF mRNA was present in the transfected MECs under the induction of prolactin, ITS and hydrocortisone. Neither nontransgenic MECs nor transgenic fibroblast cells can express hLF cDNA of 2,136 bp even with a hormone signal ( Figure 3A). Western blot also showed that the recombinant hLF was present in the supernatants of transfected MECs with the inductive medium, but not in the transfected fibroblasts and non-inductive MECs, which further confirmed mammary-specific expression of hLF transgene. However, the recombinant hLF was of a smaller mass (about 42 kDa) than standard hLF (80 kDa) ( Figure 3B).

Production of hLF Transgenic Cloned Goats
In total, 240 embryos were transplanted into 23 local recipient goats. Pregnancy was assessed on a continuous basis throughout gestation. Recipients were examined every morning and evening with healthy adult male goats. Recipients showing estrus performance were regarded as non-pregnancy and used as recipients for embryo transfer later. The recipients which did not show estrus until 40 days after embryo transfer were regarded as pregnant. In this study, 5 of 23 (21.7%) pregnancies were established on day 40 after embryo transfer. Three of them went full term. The birth body weight of cloned offspring was 2.70, 2.75, and 2.95 kg, respectively. Two pregnancies were lost around 140 days of gestation. The ears were collected for the following experiments. The lived cloned goats and aborted fetus were all females, which was consistent with the gender of donor cells.

Identification of Cloned Transgenic Status for All Clones
The five cloned goats showed the same alleles for 7 of the 8 Microsatellite loci as the donor cells (Table 1), but only showed the same alleles for one locus as the recipient goat, which might be a coincidence. These results confirmed the clone status of five transgenic goats.
Southern blot on genomic DNA (using probes specific for hLF and b-casein genes) produced fragments of the expected size for both lived and dead cloned goats (Figure 4, Figure S2). This suggested that all 5 goats were transgenic.

DNA Methylation Status of H19 DMR
The putative DMR of H19 was located upstream of the promoter, between 1,321 and 1,683 nucleotides (nt) ( Figure 5, GeneBank accession no. EF577239.1). This DMR showed 93% identity with that of sheep (GeneBank accession no. AJ566210). Also, this DMR contained one minimal CTCF (zinc-finger protein putative binding site) motif (CCGNNGGNGGC) [21], and regarded as CTCF III according to the position.

Discussion
In the present study, we firstly obtained 5 hLF transgenic cloned goats by an effective and reliable procedure. Three of the goats were birth lived, while another two died in gestation. Therefore, we further investigated the underlying epigenetic molecular mechanism of regulating the development of cloned animals by studying the DNA methylation of two imprinted genes (H19, IGF2R). Hyper-methylation of H19 and IGF2R genes was observed in CA goats, while DNA methylation in CL goats was not significantly different from NL goats.
MECs have been widely used as a model for studying the regulation of milk protein gene expression [22]. So we isolated primary MECs from Saanen dairy goats at lactating phase. MECs were used to examine the activity of hLF expression cassette of plasmid. Consistent with previous studies on transgenic expression of MEC lines in other species [23,24], the expression of recombinant hLF could be detected (Figure 3). The result confirmed that the activity of hLF expression cassette was effective for generation of hLF transgenic cloned goats. The differences in size of recombinant protein and natural protein have been widely observed. The mechanism is still not fully understood yet. In van Berkel et al. [25] and Peng et al. [26], the relative molecular mass of recombinant hLF was about 1-2 kDa smaller than that of natural hLF. This has been probably attributable to the differences in N-linked glycosylation. In the present study, the molecular weight of recombinant hLF was also smaller, which may be because the transgenic hLF was generated by the different patterns of post-translational modifications such as phosphorylation, glycosylation, sulfation, and ubiquitination between human and MECs.
The source of donor cells is an important factor affecting the efficiency of NT. Somatic cells have been mainly contributed to production of transgenic livestock, because embryonic stem cell (ESC) lines have not been obtained yet. Induced pluripotent stem cells (iPSC) are only limited to the studies in laboratory at the present stage [27,28]. The fetal fibroblast cells are the main source of donor cells for SCNT. There are still some proposed disadvantages of using fetal fibroblast cells such as comprehensive sex identification, unknown cell source, animal ethnics, high aborted percentage of offspring [10,29,30]. Lived clones obtained from adult somatic cells in sheep [31,32], cattle [33][34][35][36], and pigs [37][38][39][40] have also been reported. Kurome et al. indicated that there was no significant difference for the effects of different donor cell sources on cloning success [41]. In the present study, we used fibroblast cells isolated from ear tissue of the two-month-old dairy goat as donor cells for SCNT. We obtained a pregnant percentage of 21.7%, which is similar to that of using fetal fibroblast cells [42]. The possible reason is that the fibroblast cells of two-month-old goats partly share the characteristic of rapid propagation and potential differentiation of fetal fibroblast cells. Also, two-monthold goats also showed the genetic merit about health. The selection of goats for donor cells from healthy goats is more reliable than from a fetus with unknown phenotype. Furthermore, isolating fibroblast cells from two-month-old goats is easier and noninvasiveness to the animals. In our lab, seven lived transgenic cloned goat doeling has been obtained using transgenic fibroblast cells from three-month-old goats [9]. In another study in our lab using transgenic fibroblast cells from two-month-old goats, 47% pregnant percentage has been attained (unpublished data). Therefore, fibroblast cells from two-month-old goats might be an alternative to produce transgenic cloned goats by SCNT.
Previous reports showed that not all of the cells that survived after antibiotic selection were transgenic because of the bystander effect (transgenic cells with the antibiotic-resistance gene can provide protection to nearby non-transgenic cells either by secretion of the gene product into the medium or by direct cellto-cell contact) [43][44][45][46]. Screen of cloned embryos for EGFP before embryo transfer can be used to improve the efficiency of transgenic animals production. However, UV light in this procedure may reduce the development and implantation percentage of transgenic cloned embryos [29]. Therefore, prescreen of donor cells using extra markers such as EGFP [29] and confirmation procedures such as cell cycle distribution and flow  cytometry analysis of EGFP expression percentage before NT to get pure transgenic cells were used in our study instead. In the present study, 34 of the 64 G418-resistant cell clones expressed EGFP. After PCR, cell cycle and flow cytometry analysis, only 19 cell clones were qualified as donor cells for SCNT. Therefore, the procedure of sequential selection of transgenic cells lines may improve the efficiency of the production of transgenic animals.
The birth body weights of the 3 lived transgenic cloned goats (2.70, 2.75, and 2.95 kg, respectively) were similar to that of normal dairy goats (3.1 kg). One of the CL goats died from Coccidiosis around 3 months of age. For the two CA goats, no apparent veterinary abnormality of development in organs was found. Recently, incomplete or non-appropriate epigenetic modification of transplanted nuclei in enucleated oocytes was proposed to be the primary cause of development failure of cloned animals Horizontal gray bars represent analyzed regions and DNA length. Horizontal rectangular bar represents the putative DMR. Vertical green line represents start site of the putative DMR, located between nt 1,321 and nt 1,683 (GenBank accession no. EF577239.1). doi:10.1371/journal.pone.0077798.g005 Figure 6. DNA methylation status of H19 DMR. Methylation status of H19 DMR in goats from normal reproduction (NL), cloned lived transgenic goats (CL) and cloned aborted transgenic goats (CA). Unfilled (white) and filled (black) circles represent unmethylated and methylated CpGs, respectively. Horizontal line of circles represent one single clone that was sequenced. Lollipop diagrams were generated by BIQ Analyzer software [35]. The data was analyzed by one way ANOVA.* (P,0.05) denotes significant differences between NL goats and CA goats, *** (P,0.001) denotes very significant differences between CL goats and CA goats. doi:10.1371/journal.pone.0077798.g006 [47]. An important epigenetic mechanism confirmed was genomic imprinting which plays an important role in the reprogramming of transplanted nuclei in the enucleated oocytes [48]. The majority of imprinted genes have roles in fetal growth and development. Both the maternal and paternal genomes are required for normal development [49]. H19, IGF2 and its receptor (IGF2R) are three frequently studied imprinted genes regulating early fetal growth, and are essential for normal development of embryos [50].
The methylation status of H19 DMR shows a semi-methylation pattern in mouse and human study [50]. Studies in mouse have demonstrated that the H19 DMR regulates the expression of both H19 and IGF2 [51]. In ovine, the CpG island surrounding the CTCF III binding motif H19 DMR is subjected to differential methylation [52]. In this study, we found four putative CTCF sites of goat H19 using CCGNNGGNGGC motif. The number is the same as the ovine [21]. The methylation status of this DMR in the CA goats was significantly higher than the NL and CL goats. There was no significant difference between NL and CL goats ( Figure 6). This was consistent with the findings in cloned mice [16], cattle [53], and pigs [12,17].
Comparative analysis of the IGF2R sequence indicated that the putative DMRs was located in intron 2, which is consistent with the finding in mouse, bovine, and human [47]. Missing of IGF2R imprinting is related with large offspring syndrome in sheep [54].  . Unfilled (white) and filled (black) circles represent unmethylated and methylated CpGs, respectively. Horizontal line of circles represent one single clone that was sequenced. Lollipopdiagrams were generated by BIQ Analyzer software [35]. The data was analyzed by one way ANOVA. **(P,0.01) denotes very significant differences between NL group and CA group, CL group and CA group, respectively. doi:10.1371/journal.pone.0077798.g008 In the present study, the methylation pattern of IGF2R DMR in the NL goats and CL goats showed similar methylated pattern. However, the CA goats showed significant hyper-methylation ( Figure 8). This is consistent with the findings in the cloned bovine [53,55] and sheep [52], but different from the findings of Su et al. [53], who showed the IGF2R in dead cloned bovine was significantly hypo-methylated compared with controls.
Combining the biological functions of H19 and IGF2R [12], the results of methylation status indicated that aberrant methylation of H19 and IGF2R in CA goats may lead to the biallelic or large variable expression of genes, then may affect the cloned embryos reprogramming, finally affect the normal development of embryo and fetus. On the other hand, the normal methylation of imprinting genes in CL goats, which allow their appropriate expression as in NL goats may indicate the normal development of cloned embryo and fetus in the gestation, which could explain the survival of CL goats. This also confirmed the importance of these two imprinted gens in the development of the fetus. Thus, the methylation status of H19 and IGF2R DMRs might be considered potentially to assess the reprogramming status of transgenic cloned animals.
In conclusion, our studies indicated that SCNT can be used to generate hLF transgenic goats. We got 3 lived transgenic goats and 2 dead during gestation. To the best of our knowledge, this is the first time that methylation pattern of imprinted genes in lived and dead transgenic cloned goats were described. We confirmed that DNA methylation of H19 and IGF2R imprinted genes in the lived transgenic cloned goats was more normal than that of dead ones. These results will help further understanding of the importance of imprinted genes during fetus development. These two imprinted genes might be considered potentially to assess the reprogramming status of transgenic cloned animals. This will contribute to simplify and improve the efficiency of SCNT. File S1 Supporting tables. Table S1, Primers for the plasmid construction and PCR identification for transgenic cloned goats. Table S2, Primers for the Microsatellite loci markers, there are total 8 pairs of primers modified by6-FAM for Microsatellite loci markers. Table S3, Cell-cycle distribution percentage in fluorescence and PCR positive donor cell clone lines after contact inhibition. (DOCX)