357 EARLY FETAL DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS GENERATED FROM FIBROBLASTS GENETICALLY MODIFIED BY piggyBac TRANSPOSITION

2015 ◽  
Vol 27 (1) ◽  
pp. 266
Author(s):  
A. Alessio ◽  
A. Fili ◽  
D. Forcato ◽  
M. F. Olmos-Nicotra ◽  
F. Alustiza ◽  
...  
Keyword(s):  
Fetal Development ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Genetically Modified ◽  
Bovine Genome ◽  
Control Group ◽  
Large Animal ◽  
Phenotypic Analysis ◽  
Fluorescence Excitation ◽  
Fetal Fibroblasts

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117–8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19 184–19 189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey's test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0 ± 17.8 v. 100.0 ± 16.1 and 2.8 ± 0.8 respectively, n = 4–7; P < 0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development. Table 1.SCNT and early fetal development of bovine fibroblasts transposed with piggyBac1 The financial support of UNRC, CONICET and ANPCyT from Argentina is gratefully acknowledged.

335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 263
Author(s):  
F. Pereyra-Bonnet ◽  
A. Gibbons ◽  
M. Cueto ◽  
R. Bevacqua ◽  
L. Escobar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetically Modified ◽  
Fold Increase ◽  
Egfp Expression ◽  
Control Group ◽  
Corpora Lutea ◽  
Exogenous Dna ◽  
Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 126
Author(s):  
X. Yang ◽  
J. Mao ◽  
E. M. Walters ◽  
M. T. Zhao ◽  
K. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Natural Science ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

6 Invitro validation of gene edited phenotypes using CRISPR-dCas9 transcriptional activators

2020 ◽  
Vol 32 (2) ◽  
pp. 127
Author(s):  
K. M. Polkoff ◽  
N. K. Gupta ◽  
J. A. Piedrahita
Keyword(s):  
Dna Sequences ◽  
Fluorescent Protein ◽  
Transcriptional Start Site ◽  
Fluorescent Microscopy ◽  
Large Animal ◽  
Transcriptional Activators ◽  
Gfp Expression ◽  
Time Investment ◽  
Donor Cells ◽  
Fetal Fibroblasts

Despite the extensive advantages of gene-edited large animals for agriculture and biomedical purposes, they represent a large monetary and time investment due to high husbandry costs, long gestation lengths, and few offspring; that is, 9 months for one calf and almost 4 months for pigs. Even with known DNA sequences before somatic cell nuclear transfer (SCNT), inserted transgenes are often not expressed as expected. Therefore, there is a need to phenotypically validate the gene modifications invitro before investing time and resources in the creation of a gene-edited large animal; however, many gene targets are tissue specific and not expressed in SCNT donor cells. In this work, we show that CRISPR-dCas9 transcriptional activators (TAs) can be used to validate functional transgene insertion in nonexpressing SCNT donor cells, in our case fetal fibroblasts. To demonstrate this concept, we first generated a DNA knockin of the H2B-GFP sequence into the porcine LGR5 locus. CRISPR/Cas9 nuclease was used to create a double-stranded break in the genomic DNA downstream of the LGR5 promoter. A homology-directed repair template plasmid containing H2B-GFP flanked by 1000bp homology arms flanking the cut site was co-transfected with the Cas9 and gRNA, and cells were seeded at low density for colony outgrowth. Colonies were genotyped by PCR and sequencing to verify successful targeted transgene integration. To test whether TAs allow for invitro validation of transgene expression, 5×105 wildtype or gene-edited fibroblasts were nucleofected (Lonza) with 500ng total of four gRNA plasmids (Addgene #43860) designed to target the 1-kb region upstream of the LGR5 transcriptional start site in combination with 500ng VP64-dCas9 (Addgene #47107). Detection of green fluorescent protein (GFP) was analysed by fluorescent microscopy followed by flow cytometry; at least 30 000 events were recorded for each treatment (Cytoflex). Our results show that GFP was detected in on average 28.6% of the gene-edited cells transfected with LGR5 TAs but not detected in gene-edited cells that were not transfected with LGR5 TAs (0%) or in wild-type cells transfected with the LGR5 TAs (0%). The experiment was repeated three times. Next, to prove that our invitro validation replicates the invivo phenotype, the gene-edited colonies heterozygous for the insertion were used for SCNT to generate piglets. Epidermal cells, which contain a population of LGR5+ stem cells, were isolated from the skin and sorted for GFP expression. The RT-qPCR results from GFP+ or GFP− cells showed the presence of LGR5 transcripts in the GFP+ cells but not GFP− cells. In conclusion, TAs were necessary and sufficient to detect LGR5-promoter driven H2B-GFP expression in gene-edited fibroblasts invitro, which faithfully recapitulates the invivo phenotype of the gene-edited animal. Further preliminary data from our laboratory suggest that our novel method can be used to detect successful gene knockouts in addition to transgene knockins and can be used to validate phenotypic outcomes of DNA modifications before the generation of gene-edited animals.

309 PRODUCTION OF CATTLE BY NUCLEAR TRANSFER FROM CELLS IN WHICH A GENE IS DISRUPTED

2009 ◽  
Vol 21 (1) ◽  
pp. 251
Author(s):  
M. Urakawa ◽  
Y. Sendai ◽  
A. Ideta ◽  
K. Hayama ◽  
Y. Shinkai ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Gene Targeting ◽  
Prion Protein ◽  
Nuclear Transfer ◽  
Antibiotic Resistance Gene ◽  
Control Group ◽  
Gestation Length ◽  
Entry Site ◽  
Donor Cells ◽  
Fetal Fibroblasts

Gene-targeted animals provide a powerful model to examine gene functionality. In this study, we examined the effect of gene targeting of donor cells for nuclear transfer (NT) on the pregnancy rate and on viability of the offspring after embryo transfer. Gene-targeted (tg; targeting of both alleles of the gene encoding bovine prion protein) or non-manipulated (control) bovine fetal fibroblasts were used for NT. A promoterless positive selection vector (pPrP5.2) containing an internal ribosome entry site-antibiotic resistance gene (neo) cassette and loxP sequences was used to disrupt the bovine prion protein gene. The cells (tg) in which homologous recombination was occurred were used for NT. The tg and control cells were cultured in DMEM with 10% FCS and were prepared in the early G1 phase to our previous report (Urakawa M et al. 2004 Theriogenology 62, 714–728). Each donor cell was inserted into an enucleated in vitro-matured (19 h) oocyte. Cell fusion (DC, 200 V mm–1, 10 μs) and activation (DC, 100 V mm–1 , 60 μs) were done in 0.3 m mannitol solution. The NT embryos were then activated with 5 μm Ca-ionophore and 10 μg of mL–1 cycloheximide and were cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were judged at 6 days after NT. The blastocysts were non-surgically transferred to recipient heifers. The recipients were monitored daily for heat behavior, examined by ultrasound at Day 30 and 60, and then observed monthly to confirm pregnancy. The offspring born in the tg group were confirmed by PCR to be transgenic. Statically significance was tested using a chi-square test or t-test. Developmental rate to the blastocyst stage, pregnancy rate at Day 30 and 60, and calving rate did not differ significantly between tg and the control group (Table 1). Gestation length (tg; 290.0 ± 2.2 days v. control; 290.5 ± 3.9 days) and birth weight (tg; 39.6 ± 8.0 kg v. control; 40.2 ± 4.1 kg) were not significantly different. These results indicate that gene targeting of donor cells used for NT does not significantly affect the development of embryos, pregnancy rate, or the viability of the offspring. Table 1.Development of NT embryos with tg or control cells

29 SCRIPTAID IMPROVES SOMATIC NUCLEAR TRANSFER EFFICIENCY DURING IN VITRO CULTURE OF PORCINE EMBRYOS DERIVED FROM INBRED MINIATURE PIG FETAL FIBROBLASTS

2015 ◽  
Vol 27 (1) ◽  
pp. 107
Author(s):  
R. Koppang ◽  
N. R. Mtango ◽  
M. Barcelo-Fimbres ◽  
J. P. Verstegen
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
Treated Group ◽  
Culture Conditions ◽  
Control Group ◽  
Miniature Pig ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Porcine somatic cell nuclear transfer (SCNT) is limited to the same or next day surgical embryo transfer due to poor culture conditions in vitro. In this study, we aimed to overcome this problem by treating SCNT embryos with scriptaid, an inhibitor of histone deacetylase (HDACi) that helps with epigenetic reprogramming of the somatic nuclei. Scriptaid was chosen over other HDACi because it has been shown to improve histone acetylation in the same pattern as that of IVF embryos as well as its low toxicity characteristic (Zhao et al. 2009 Biol. Reprod. 81, 525–530; Zhao et al. 2010 Cell Reprogram. 12, 75–78). An inbred miniature pig fetal fibroblast cell line that is known to give low blastocyst rate in culture was used as a source of donor cells transferred into enucleated oocytes. Traditional SCNT was performed; embryos were fused and chemically activated in 10 µM ionomycin for 5 min and 2 mM DMAP for 3 to 4 h before being transferred into scriptaid. Embryos were treated with 500 nM scriptaid (Zhao et al. 2010) for 18 h and the untreated group was used as control. A total of 806 oocytes were used in 8 replicates. The constructed embryos were cultured in modified porcine zygote medium 5 (mPZM-5) for 7 days at 39°C in 5% O2, 5% CO2, 90% N2 atmosphere. Cleavage rates were assessed at 2.5 days and blastocyst rates at Day 7 after activation. Data were analysed by ANOVA using GLM, and percentages were transformed using arcsin square root using Statistix 10 software (Tallahassee, FL, USA). There were no differences in cleavage rates for control group v. scriptaid (55.3 v. 49.9%; P > 0.1; Table 1). The blastocyst rate per construct showed remarkable increase in the scriptaid treated group compared with the control group (12.8 v. 2.2%; P < 0.01; Table 1). Similarly, a significant effect was observed for blastocyst per embryos cleaved where scriptaid had higher rates compared with control (25.8 v. 5.8%; P < 0.01). These results indicated that improving nuclear reprogramming of miniature porcine SCNT clones by scriptaid treatment enhanced blastocyst production during the in vitro culture of porcine embryos. Table 1.Mean (± s.e.m.) measures of embryonic development of SCNT embryos

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

2000 ◽  
Vol 12 (2) ◽  
pp. 1 ◽  
Author(s):  
Sangho Roh ◽  
Hosup Shim ◽  
Woo-suk Hwang ◽  
Jong-taek Yoon
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

scholarly journals Transcriptome-wide analysis of the SCNT bovine abnormal placenta during mid- to late gestation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Guangqi Gao ◽  
Shenyuan Wang ◽  
Jiaqi Zhang ◽  
Guanghua Su ◽  
Zhong Zheng ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Fetal Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Late Gestation ◽  
Differentially Expressed ◽  
Transmembrane Transport ◽  
Control Group ◽  
Genomic Information ◽  
Competing Endogenous Rnas

AbstractThe dysfunction of placenta is common in somatic cell nuclear transfer (SCNT) cloned cattle and would cause aberrant fetal development and even abortion, which occurred with highest rate at the mid- to late gestation. However, the mechanism of abnormal placentas was unclear. To analyze the transcriptome-wide characteristics of abnormal placentas in SCNT cloned cattle, the mRNA, lncRNA and miRNA of placental cotyledon tissue at day 180 after gestation were sequenced. A total of 19,055 mRNAs, 30,141 lncRNAs and 684 miRNAs were identified. Compared with control group, 362 mRNAs, 1,272 lncRNAs and nine miRNAs (six known and three novel miRNAs) were differentially expressed (fold change ≥ 2 and P-value < 0.05). The differentially expressed genes were functionally enriched in urea and ions transmembrane transport, which indicated that the maternal-fetal interactions were disturbed in impaired placentas. Furthermore, the competing endogenous RNAs (ceRNAs) networks were identified to illustrate their roles in abnormal placental morphology. The present research would be helpful to discover the mechanism of late gestational abnormality of SCNT cattle by provides important genomic information and insights.

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