Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats

Theriogenology 81 (2014) 459–466

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Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats Li Meng a,1, Ruo-Xin Jia a,1, Yan-Yan Sun b, Zi-Yu Wang a, Yong-Jie Wan a, Yan-Li Zhang a, Bu-Shuai Zhong a, Feng Wang a, * a b

Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing, PR China Animal Breeding and Genomics Centre, Wageningen University, Wageningen, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 August 2013 Received in revised form 20 October 2013 Accepted 22 October 2013

Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung development. Our objective was to investigate molecular background of lung developmental problems in transgenic (random insertion of exogenous DNA) cloned goats. We examined expression of 15 genes involved in growth regulation, imprinting, and epigenetic transcription in lung tissue of deceased transgenic cloned and normal goats of various ages. Compared with normal goats of the same age from conventional reproduction, expression of 13 genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, CDKNIC, PCAF, MeCP2, HDAC1, and Dnmt3b) decreased in transgenic cloned goats that died at or shortly after birth; Expression of eight genes (FGF10, PDGFR, RABP, VEGF, PCAF, HDAC1, MeCP2, and Dnmt3b) decreased in fetal death of transgenic cloned goats. Expression of two epigenetic transcription genes (PCAF and Dnmt3b) decreased in disease death of transgenic cloned goats (1–4 months old). Disruptions in gene expression might be associated with the high neonatal mortality in transgenic cloned animals. These findings have implications in understanding the low efficiency of transgenic cloning. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Growth-regulation Imprinting Epigenetic transcription Transgenic cloned goat qRT-PCR

1. Introduction Somatic cell nuclear transfer (SCNT) [1,2], which uses preselected genetically modified cells as donor nuclei [3], opens new horizons for application of transgenic technologies in livestock animals. The production of the first transgenic cloned goat using SCNT [4] came after reports of the first transgenic cloned sheep, cow, and mouse, and was followed by cloning of many other large animal species. Cloning of other species can reach the efficiency up to 4% (total live kids/embryos transferred), whereas average * Corresponding author. Tel.: þ86 25 84395381; fax: þ86 25 84395314. E-mail address: [email protected] (F. Wang). 1 These two authors are co-first authors. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.10.023

efficiency in goats is only 2.6% [5]. The low overall success rate is a cumulative result of inefficiencies at each stage of the process, including embryonic, fetal, prenatal, and neonatal loss, and production of abnormal offspring. Initial and subsequent SCNT-derived large animals including goats, furthermore, still have a lot of health issues and a shortened life span [6,7]. The primary reason for low efficiency of cloning and health problems in cloned animals is incomplete reprogramming of donor somatic cell nuclei, which might lead to abnormal or even lack of expression of some important growth regulation, imprinting, and epigenetic transcription-related genes [8–11]. Deviations in expression of these genes, furthermore, are known to lead to phenotypic abnormalities of cloned animals [12,13]. The

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deregulation of one or more key gene(s) crucial for development could result in dying of the embryo at each stage of embryo development. Some cloned embryos with some deregulation of gene expression in lungs could still develop to birth [14], however, would still die of lung abnormality after birth. Some cloned cattle produced to date also showed abnormal development in lungs [15]; transgenic cloned goats, similarly, showed abnormal respiratory symptoms and anatomical abnormalities in lungs in our previous studies (unpublished data). The molecular background of respiratory problems of transgenic cloned goats, however, remains elusive. Our objective in the present study was, therefore, to investigate gene expression patterns in lungs of transgenic cloned and normal goats, which might reveal possible genetic causes of death of transgenic cloned goats. We selected 15 genes to conduct real-time polymerase chain reaction (PCR) and these genes functioned in growth regulation, imprinting, and epigenetic transcription. For growth regulation genes, vascular endothelial growth factor (VEGF) [16], platelet-derived growth factor receptor (PDGFR) [17], bone morphogenetic protein 4 (BMP4) [18,19], fibroblast growth factor 10 (FGF10), retinoic acid-binding proteins (RABP), hepatocyte growth factor receptor (HGFR), and growth hormone receptor (GHR) are involved in embryo development and organogenesis. For imprinting genes, X-inactive specific transcript (Xist) plays a crucial role during X-chromosome inactivation [20]. H19, insulin-like growth factor 2 receptor (IGF2R), and cyclin-dependent kinase inhibitor (CDKNIC) play significant roles in embryo development and organogenesis [10,21]. For epigenetic transcription factor genes, P300/CBP (cAMP-response-element-binding protein [CREB]-binding protein) associated factor (PCAF) participates in transcriptional activation, cell-cycle arrest, DNA damage response, and apoptosis [22]. DNA methylation CpG-binding protein-2 (MeCP2) and histone deacetylase 1 (HDAC1) [23] play roles in inaccessibility of transcription factors to bind to DNA. Finally, de novo methyl transferase (Dnmt3b) functions in establishing DNA methylation patterns during development [24]. 2. Materials and methods 2.1. Animals The deceased hLF transgenic cloned goats were collected from previous studies that produced transgenic cloned goats using SCNT [25]. Briefly, goat fibroblast cells from ears of 3-month-old goats were used to produce donor somatic cells harboring the hLF gene for SCNT. Culture and passage of donor cells, oocyte collection and enucleation, nuclear transferring, oocyte activation, embryo culture, and embryo transfer were carried out in order [25]. Normal goats of the same age were from conventional reproduction as control goats. The transgenic cloned goats and six normal goats, with the same genetic background, were housed in the same husbandry conditions. The obtained hLF transgenic cloned goats were divided into three groups based on the age of death: (1) fetus death group (FTC) that were produced as death before expected birth date, including three transgenic cloned goats named FTC1, FTC2, and FTC3; (2)

newborn death group (NTC), that died at or shortly after birth, including three transgenic cloned goats named NTC1, NTC2, and NTC3; and (3) 1- to 4-month-old death group (OTC), including three transgenic cloned goats named OTC1, OTC2, and OTC3, that deceased at 1, 2, and 4 months of age, respectively. The transgenic cloned goats of 2 and 4 months of age died of pneumonia and coccidiosis, respectively. The death cause of the 1-month-old transgenic cloned goat was not clear. For the FTC and NTC groups, three newborn normal goats from conventional reproduction were used as control goats. For the OTC group, three normal goats from conventional reproduction that were 1, 2, and 4 months of age, respectively, were used as control goats. 2.2. Tissue collection The apex of lung tissues were collected immediately after death of transgenic cloned goats, or killing of normal control goats using overdose with barbiturate. The lung tissues were snap frozen in liquid nitrogen and stored at 80  C until further use. Animal handling and experimentation were in accordance with the National Research Council’s publication “Guide for the Care and Use of Laboratory Animals” and approved by Institutional Animal Care and Use Committees at Nanjing Agricultural University. 2.3. Preparation of RNA Total RNA was extracted from lung samples of transgenic cloned goats and control goats using an RNAEASY kit (Qiagen), following manufacturer’s instructions. RNA quality was confirmed using ratios of A260/A280 and A260/A230 (Nanodrop, all between 1.8 and 2.0) and also using automated electrophoresis (BioRad Experion) for the presence of three clear ribosomal RNA bands. 2.4. Reverse transcription Reverse transcription (RT) was done using an RT kit (Roche) with approximately 1 mg of RNA in a total volume of 20 mL. The RT reaction was conducted following manufacturer’s guidelines using OligodT primers and avian myeloblastosis virus reverse transcriptase enzyme in a volume of 20 mL to prime the RT reaction and produce cDNA. 2.5. Quantitative real-time PCR Quantitative real-time PCR was performed using ABI Prism 7300 Sequence Detection. The PCR reaction consisted of 12.5 mL of SYBR Green PCR Master Mix (Applied Biosystems), 300 nM of forward and reverse primers, and 2.0 mL of 1:20-diluted template cDNA, in a total volume of 25 mL. The primers used in real-time PCR are shown in Table 1. Cycling was performed using default conditions of ABI Prism 7300 SDS Software 1.0: 2 minutes at 50  C, 10 minutes at 95  C, 40 rounds of 15 seconds at 95  C, and 1 minute at 60  C. To verify that one pair of primers produced only one single product, a dissociation protocol was added after thermocycling, determining dissociation of PCR products from 65  C to 95  C. A standard curve using serial dilutions of pooled sample (cDNA from all samples), a

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Table 1 Primers for quantitative real-time PCR. Gene

Primer sequence (50 –30 )

Product size (bp)

Sequence accession

Annealing temperature ( C)

Ea

Dnmt3b

F-GCCTTCCTGTAAGAGACCAGCTT R-TGGTGGCATTGGGACTGTT F-ATTGACGACGAGTCCTATGAG R-ATGCCCTTTGATGGTCAGATTG F-CCGCTGGGAAGTATGATGTG R-GGGAGATTTGGGCTTCTTAGG F-GAACTTTCTGCTCTCTTGGG R-CTGGCTTTGGTGAGGTTTGA F-CTTTTCGTTTCCTCTTTAACC R-TCCCACCGTGTCACATTGT F-AACTCCTCTTCTTCTTCTTCCTC R-TTCACAGCAACAACTCCGAT F-GAAGTATCAGCTTCCCAACTTC R-AAACAATCTGGGTGTTCC F-CGGCTCGCAGGTCCTACAGGT R-GGTGAACGGCACTTGGTGAAT F-GAGTGAAGTGAGCTGGCAGT R-TCCGTCATTCCTAGAGGTAC F-TTTCTGTCAGCACACTCGGC R-CAAGGGTTTTGTGTTTCGGG F-CCCACTTGGGAGAATGAGAA R-GCCTTCATTCCCGAACATAA F-GCCCATCTAGCTTGCAGTCTCT R-CAGACGGCTCAGGAACCATT F-AACCTCACGCCATTCCTCTG R-GGGTAGGTGTTCCTCTTGAG F-GGACTGGAACTTGGACTTCTTCA R-TGGTGTGGGTCTTCCGTTC F-CCAGCGTCTGTGACTTCGTG R-CCATCCTTGCAGCCTCCTTC F-CGACTTCAACAGCGACACTCAC R-CCCTGTTGCTGTAGCCCAATTC

330

NM_181813

58

1.94

150

BC108088

55

1.86

191

XM_588477

55

1.91

340

NM_174216

61

1.80

229

AF136233

54.5

1.89

239

AY183659

58

1.83

171

NM _000245

56.5

1.81

197

NM _176608

61

1.87

249

NM _033023

62

1.84

220

NM_003884

61

1.83

166

M17253

54.5

1.84

112

NM_001142510

56

1.86

226

AF104906

56

1.86

133

AY091484 (sheep)

55.5

1.93

220

DQ666954

60

1.87

118

NM_001034034

58

1.88

HDAC1 MeCP2 VEGF BMP4 FGF10 HGFR GHR PDGFR PCAF RABP CDKNIC Xist H19 IGF2R GAPDH

Abbreviations: bp, base pair; E, amplification efficiency; F, forward; PCR, polymerase chain reaction; R, reverse. a Amplification efficiency derived from the formula E ¼ 10(1/slope).

negative control without cDNA template, and a negative control without reverse transcriptase were taken along with each assay. For each pair of primers, efficiency curves were generated using serial dilutions of cDNA in abscissa and corresponding cycle threshold (Ct) in ordinate. The slope of log-linear phase reflects amplification efficiency (E) derived from the formula, E ¼ 10(1/slope). Amplification efficiency obtained for each primer pair is indicated in Table 2. For quantification analysis, Ct of the target gene was compared with the internal reference gene GAPDH according to the ratio, R ¼ (EGAPDHCt GAPDH/EtargetCt target) expressed as a percentage [26]. 2.6. Statistical Analysis An independent sample t test was used to analyze differences of mRNA expression from quantitative real-time PCR between transgenic clones and normal control goats using SAS. Differences were considered to be statistically significant at P < 0.05. 3. Results 3.1. Lung morphology of transgenic cloned goats In the FTC group, atelectasis accompanied with thickening of the alveolar wall was observed in all three

transgenic cloned goats. In the NTC group, atelectasis accompanied with thickening of the alveolar wall was observed in lungs of NTC2 and NTC3. In the OTC group, OTC2 showed congestion and inflammation in the lung, and for other transgenic cloned goats, no apparent Table 2 Principal abnormalities at necropsy of deceased transgenic cloned goats. Animal

Birth weight, kg

Lung development status

Age at death, days

Cause of death

FTC1

No data

B 140

FTC2

No data

FTC3

No data

NTC1

3

Atelectasis, thicken alveolar wall Atelectasis, thicken alveolar wall Atelectasis, thicken alveolar wall Normal

NTC2

2.7

P3

NTC3

4.4

P2

ND

OTC1 OTC2

2.75 2.95

P 145 P 61

OTC3

1.5

Atelectasis, thicken alveolar wall Atelectasis, thicken alveolar wall Normal congestion and inflammation Normal

Naturally aborted Naturally aborted Naturally aborted Intestine disease ND

Coccidiosis Inflammation of lung ND

B 138 B 120 P3

P 29

Abbreviations: B, before birth; FTC, fetus transgenic clone; ND, not determined; NTC, newborn transgenic clone; OTC, 1- to 4-month-old transgenic clone; P, after birth.

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veterinary abnormality of development was found in the lungs (Table 2). 3.2. Expression level of growth-regulation genes Gene expression in transgenic cloned goats was first compared with normal goats of the same age from conventional reproduction. We studied the expression of seven growth-regulation genes. In the FTC group, the expression level of FGF10 (P < 0.01), PDGFR (P < 0.05), RABP (P < 0.01), and VEGF (P < 0.05) was reduced compared with that of control goats. BMP4, GHR, and HGFR showed more variation in expression level in transgenic cloned goats compared with the control group. Individual transgenic clones showed extremely low gene expression levels (Fig. 1). For example, expression of BMP4 was approximately five times lower in FTC1 and FTC2 compared with control goats. The expression of GHR, furthermore, was 12 times reduced in FTC1 and eight times reduced in FTC2 compared with control goats. In the NTC group, the expression level of BMP4 (P < 0.01), FGF10 (P <

0.01), GHR (P < 0.01), HGFR (P < 0.01), PDGFR (P < 0.05), RABP (P < 0.01), and VEGF (P < 0.05) was decreased compared with control goats. In the OTC group, no difference was found in expression of growth-regulation genes compared with control goats. The expression levels of BMP4 (P < 0.05), FGF10 (P < 0.01), GHR (P < 0.05), HGFR (P < 0.01), RABP (P < 0.05), and VEGF (P < 0.01) were greater in newborn control goats than in 1to 4-months-old control goats, highlighting the role of those genes in lung development. 3.3. Expression level of imprinting genes We studied four imprinting genes (H19, IGF2R, Xist, and CDKNIC). The expression of H19 (P < 0.05) and CDKNIC (P < 0.01) was different in the NTC group compared with the control goats. In the FTC group, expression variance of four genes was greater than that of control goats, although the difference was not significant. Individual clones showed extremely high or low expression (Fig. 2). For instance, FTC3 had seven

Fig. 1. Expression of growth regulation genes in three groups of deceased transgenic cloned and normal control goats. For FTC and NTC in the dead clone groups, the control is NTC in the control group; for OTC in the dead clone groups, the control is OTC in the control group. * P < 0.05; ** P < 0.01; *** P < 0.001. BMP4, bone morphogenetic protein 4; FGF10, fibroblast growth factor 10; FTC, fetus transgenic clone; GHR, growth hormone receptor; HGFR, hepatocyte growth factor receptor; NTC, newborn transgenic clones; OTC, 1- to 4-month-old transgenic clone; PDGFR, platelet-derived growth factor receptor; RABP, retinoic acid-binding proteins; VEGF, vascular endothelial growth factor.

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Fig. 2. Expression of imprinting genes in three groups of deceased transgenic cloned and normal controls goats. For FTC and NTC in the dead clone groups, the control is NTC in the control group; for OTC in the dead clone groups, the control is OTC in the control group. * P < 0.05; ** P < 0.01. CDKNIC, cyclin-dependent kinase inhibitor; FTC, fetus transgenic clone; IGF2R, insulin-like growth factor 2 receptor; NTC, newborn transgenic clone; OTC, 1- to 4-month-old transgenic clone; XIST, X-inactive specific transcript.

times more CDKNIC gene expression than average expression levels of control goats. In the OTC group, expression of H19 and CDKNIC were relatively uniform in control and transgenic cloned goats. Although no significant changes were found in expression of IGF2R between transgenic cloned and control goats, OTC2 had 15 times more IGF2R gene expression, and OTC3 had seven times more than average expression levels of control goats. IGF2R and H19 expression was greater in neonatal control goats than in 1- to 4-month-old old control goats, reflecting the need for a greater amount of these two growthimprinting genes during the postnatal growth period. 3.4. Expression level of epigenetic transcription-related genes PCAF, HDAC1, MeCP2, and Dnmt3b (Fig. 3) were investigated to check the expression level of epigenetic regulation factors of transcription in the FTC, NTC, and OTC groups. PCAF, MeCP2, HDAC1, and Dnmt3b all showed different expression (P < 0.01) in the FTC and NTC groups compared with control goats. In the OTC group, expression of PCAF and Dnmt3b were found to be abnormally lower than that in control goats. The expression of PCAF, HDAC1, MeCP2, and Dnmt3b were greater in neonatal control goats than in 1- to 4month-old control goats, reflecting a need for a greater amount of these epigenetic regulation factors of transcription genes during the postnatal growth period. 4. Discussion To determine possible genetic causes of death in transgenic cloned goats from SCNT, expression levels of 15

growth regulation, imprinting, and epigenetic transcription-related genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, IGF2R, Xist, CDKNIC, HDAC1, MeCP2, PCAF, Dnmt3b) were compared in lungs of deceased transgenic cloned goats with normally reproduced control goats using quantitative real-time PCR. These genes are known to have important functions during the development of the embryo and organogenesis in mammals. Seven growth regulation genes, two imprinting genes, and three transcription regulation factor genes were found to be reduced in the NTC group compared with the control goats. Five growth regulation genes and three transcription regulation factor genes had a reduced expression in the FTC group compared with the control goats. Only two epigenetic transcription-related gene expression levels, however, were reduced in the OTC group compared with control goats. BMP4 has an important role in regulation of epithelial proliferation and proximal-distal cell fate during lung morphogenesis [18,19]. BMP4 showed a lower expression level in lungs of NTC goats compared with control goats. Aberrant morphogenesis was also found in lungs of some clones. Both NTC2 and NTC3 showed very poor lung morphosis-atelectasis, accompanied by thickening of the alveolar wall. Overexpression of BMP4 caused abnormal lung morphogenesis, with cystic terminal sacs and inhibition of epithelial proliferation [9]. Therefore, aberrant expression of BMP4 might contribute to lung abnormalities and death of the NTC group. GH regulates a wide range of biological processes and is mediated by GHR [27]. GH combined with GHR is normally involved in early lung growth, oxidative protection, lipid, and energy metabolism, and in proteasomal activity by GH signaling [28]. Hepatocyte growth factor (HGF) is mediated by HGFR, elicits proliferation, motility, differentiation, and

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Fig. 3. Expression of epigenetic transcription-related genes in three groups of deceased transgenic cloned and normal controls goats. For FTC and NTC in the dead clone groups, the control is NTC in the control group; for OTC in the dead clone groups, the control is OTC in the control group. * P < 0.05; ** P < 0.01; *** P < 0.001. Dnmt3b, de novo methyl transferase; FTC, fetus transgenic clone; HDAC1, histone deacetylase 1; MeCP2, DNA methylation CpG-binding protein-2; NTC, newborn transgenic clone; OTC, 1- to 4-month-old transgenic clone; PCAF, P300/CBP (cAMP-response-element-binding protein [CREB]-binding protein)-associated factor.

morphogenesis of epithelia and other cells, and is essential for embryonic development [29,30]. Aberrantly lower expression of GHR and HGFR might also have contributed to abnormal development of lungs and death in the NTC group. FGF10 is responsible for directed outgrowth of lung endoderm [31]. FGF10/ mice showed multiple organ defects including lung agenesis [32]. PDGFR/ mice that survive birth develop a lung emphysema-like condition reflecting a complete failure of alveolar septum formation [33]. PDGFR combined with PDGF could promote proliferation (and possibly also migration and differentiation) of specific populations of mesenchymal cells that drive morphogenesis of lung alveolar septa and intestinal villi [17]. In the present study, lower expression of FGF10 and PDGFR might contribute to lung defects as reported in the NTC group and death in the FTC group. In the FTC, NTC, and OTC groups, a lower expression of PCAF was observed compared with control goats. Therefore, transgenic cloning might affect normal expression of PCAF. Retinoic acid (RA) signaling that is mediated by nuclear receptor retinoic acid receptor and RABP [14], is required for lung bud initiation and subsequent branching morphogenesis in lung and RA signaling. The VEGF family and their receptors are key mediators of vascular growth and remodeling in a variety of tissues, including human endometrium [16]. Endometrial secretions, including VEGF, play important roles during embryo implantation [34]. VEGF receptor signaling inhibition [35] or disruption of capillary endothelial cell organelles [36] results in abnormal lung vascular growth and reduced valvulogenesis. Lower expression of RABP and VEGF was found in the FTC and NTC groups, which might also contribute to lung abnormalities of neonatal transgenic cloned goats and death of goats in the two groups.

Both DNA methylation and histone acetylation on nuclear chromatin are involved in regulation of gene activation, where histone tails mediated chromatin remodeling [37]. Methylated DNA binds transcriptional repressor MeCP2 that recruits HDACs [26]. Ultimately, HDACs remove acetyl groups from histones H3 and H4, altering chromatin configuration, which further supports inaccessibility of transcription factors to bind to DNA sequences [38]. PCAF, a transcriptional coactivator with intrinsic histone acetylase activity, plays roles in transcriptional activation, cell-cycle arrest, DNA damage response, and apoptosis, and contributes to transcriptional activation by modifying chromatin and transcriptional factors [22,39]. Dnmt3b establishes DNA methylation patterns during development, missense mutations cause immunodeficiency, centromere instability, and facial anomalies syndrome [24]. In the present study, the abnormal lower expression level of MeCP2, HDAC1, PCAF, and Dnmt3b genes were found in both the FTC and NTC groups. Aberrantly lower expression of PCAF, and Dnmt3b was also found in the OTC group. These results suggest that transgenic cloning probably leads to aberrant epigenetic activity affecting normal expression MeCP2, HDAC1, PCAF, and Dnmt3b of lung of transgenic cloned goats. It is accepted that SCNT is an inefficient process, and that the developmental anomalies during cloning might be caused by incomplete epigenetic reprogramming of the donor nucleus, which might cause dysregulation of imprinting gene expression [40,41]. There are many studies on roles of imprinting genes in fetal development and placental differentiation, growth, and function [42]. CDKNIC is a paternally imprinting gene and in most normal tissues is expressed from a maternal allele although frequently leaky expression also occurs from paternal allele [21,43].

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CDKNIC has recently been shown to be a critical regulator of embryonic growth and is essential for normal development [44]. Both IGF2R and H19 involved in fetal growth regulation are paternally imprinting, characteristically maternally expressed genes that encode a growth-inhibitory factor and nonprotein-coding RNA transcript of unknown function, respectively, and are essential for animal development [10]. Xist is a large noncoding RNA, exclusively transcribed from the inactivated X chromosome of female mammals [45]. Xist plays an important role in gene repression in the early embryo, and later stabilizes the inactive state [46]. The expression of CDKNIC and H19 was reduced in the NTC group compared with control goats. Despite that no significant difference was found in IGF2R and Xist gene expression between NTC and control goats, individual clones showed extremely aberrant viable expression. Lung organs in the FTC group were also more variable in expression of these four genes compared with control goats. These observed highly variable results might reflect a very low efficiency of reprogramming in lungs of the FTC and NTC groups. This might explain aberrant lung development of the NTC group and death of two groups. Although several transgenic cloned goats were derived from the same donor cell line and were therefore genetically identical, they showed larger variation in some gene expression levels, especially for imprinting genes (IGF2R and Xist) in all three groups. A larger variation (BMP4, GHR, HGFR, IGF2R, H19, Xist, and CDKNIC) was observed in the FTC group than in the other two groups. This is consistent with reports from numerous other groups in phenotypic parameters [47] and gene expression [12,13,48] of cloned pig, cattle, and mice. This indicated that aberrant expression of these genes hampered the normal development process, which might further have lead to death in the FTC group. In contrast to the FTC and NTC groups, transgenic clones that survived to 1 to 4 months of age, even those that suffered diseases and died eventually, appear to be more or less normal in expression of genes studied, except for PCAF and Dnmt3b. This result was consistent with gene expression in cloned cattle that survived [13], indicating that transgenic cloned goats that survived to 1 to 4 months of age are relatively normal at the molecular level of those genes. Expression levels of 12 genes were greater in neonatal control goats compared with goats with 1- to 4month-old control goats. The expression level difference of imprinting genes between neonatal and adult animals have also been reported in calves [13], and reports about growth regulation genes and epigenetic transcription factors between these two ages have not been seen yet. This result reflected that the need of neonatal animals for a greater amount of these important development regulation genes during the postnatal growth period. Limited numbers of transgenic goats created via SCNT were used in our study because of the low production efficiency of transgenic cloning. The observed expression differences of the genes of interest between deceased transgenic cloned goats and control goats, however, might facilitate our understanding of molecular mechanisms responsible for developmental abnormalities of transgenic cloned animals. These findings might also contribute to further development of animal cloning technology. We

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believe this is the first time that growth regulation, imprinting, and epigenetic transcription-related gene expression in transgenic cloned goats have been described. 4.1. Conclusions Aberrant expression of growth regulation genes, imprinting genes, and epigenetic transcription factors was observed in both FTC and NTC goats, and expression appeared to be normal in 1- to 4-month-old transgenic cloned goats. Transgenic cloning techniques might cause aberrant epigenetic nuclear reprogramming and exogenous gene insertional mutagenesis or ectopic expression, which might disrupt normal developmental regulation of the fetus. The abnormal development of the fetus might result in developmental insufficiencies, and ultimately, fetal or perinatal death in transgenic cloned goats. These findings have implications in understanding low efficiency of transgenic cloning. Acknowledgments The authors greatly appreciate Loes P.M. Duivenvoorde for carefully editing and correcting the manuscript. The authors thank Guo-Min Zhang for production of transgenic cloned goats, Xue-Qiong Wang for helping with performance of real-time PCR. This study was financially supported by National Major Special Projects on New Cultivation for Transgenic Organisms (Number 2013ZX08008-004), Jiangsu Povincial Agricultural Supporting Program (BE2010372), and Fundamental Research Funds for the Central Universities (No.KYZ201211). References [1] Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. Fullterm development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998;394:369–74. [2] Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, et al. Somatic cell nuclear transfer. Nature 2002;419:583–6. [3] Galli C, Lagutina I, Perota A, Colleoni S, Duchi R, Lucchini F, et al. Somatic cell nuclear transfer and transgenesis in large animals: current and future insights. Reprod Domest Anim 2012;47(Suppl 3): 2–11. [4] Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, et al. Production of goats by somatic cell nuclear transfer. Nat Biotechnol 1999;17:456–61. [5] Boulanger L, Passet B, Pailhoux E, Vilotte JL. Transgenesis applied to goat: current applications and ongoing research. Transgenic Res 2012;21:1183–90. [6] Watanabe S, Nagai T. Survival of embryos and calves derived from somatic cell nuclear transfer in cattle: a nationwide survey in Japan. Anim Sci J 2011;82:360–5. [7] Watanabe S, Nagai T. Death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer and their progeny: a result of nationwide survey in Japan. Anim Sci J 2009;80:233–8. [8] Su JM, Yang B, Wang YS, Li YY, Xiong XR, Wang LJ, et al. Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves. Theriogenology 2011;75:1346–59. [9] Li S, Li Y, Du W, Zhang L, Yu S, Dai Y, et al. Aberrant gene expression in organs of bovine clones that die within two days after birth. Biol Reprod 2005;72:258–65. [10] Shen CJ, Cheng WT, Wu SC, Chen HL, Tsai TC, Yang SH, et al. Differential differences in methylation status of putative imprinted genes among cloned swine genomes. PLoS One 2012;7:e32812.

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