Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other...Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.展开更多
The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, method...The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout (KO) cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.展开更多
Factors affecting the efficiency of nuclear transfer (NT) in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted ...Factors affecting the efficiency of nuclear transfer (NT) in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses (P〈 0.05), but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups (P〉0.05). When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos (P〈 0.05), although the fusion rate did not significantly differ among the three groups (P〉 0.05). Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage (P 〈 0.05), although the fusion rate was similar (P 〉 0.05). Activation of cytoplasts prior to fusion increased the cleavage rate (P〈 0.05) and blastocyst development (P〈 0.05) of reconstructed embryos, but decreased the fusion rate (P 〈 0.05) compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM + 3% OCS at the first 48 h and then cultured in TCM199+ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199+ 10% FCS or TCM199+3% OCS (P 〈 0.05). When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.展开更多
The experiments of serial nuclear transfer were conducted between Boer goat and rabbit. The enucleated oocytes of rabbit were used as recipients while the blastomeres of goat morula was used as nuclear donor. The reco...The experiments of serial nuclear transfer were conducted between Boer goat and rabbit. The enucleated oocytes of rabbit were used as recipients while the blastomeres of goat morula was used as nuclear donor. The reconstructed embryos developing to morula were used as donor for serial cloning. As a result, two generations of reconstructed embryos were obtained, including 58 first generation reconstructed embryos and 14 second generation reconstructed embryos. The fusion rates were 79.5 and 70%, respectively, and there was no significant difference between them (P〉0.05). The cleavage rates were 75.9 and 28.6% respectively with significant difference (P〈0.01). No blastocyst was obtained from the second generation reconstructed embryos while 13.8% of first generation reconstructed embryos developed to blastocyst.展开更多
To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the ...To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.展开更多
This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (St...This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.展开更多
Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole)...Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and electroactivation.According to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:1.thosewith chromosomes juxtaposed to the polar body;2.those with chromosomes in its adjacency; and3.those with chromosomes further removed.The relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further on.In addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 hours.As aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 hours.By so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.展开更多
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover,...Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.展开更多
Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryo...Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two(P<0.05).The development rate of embryos reconstruct-ed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization(FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that(1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA genotype through FISH.展开更多
Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors (WHO). Athero- sclerosis is a chronic in...Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors (WHO). Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall (Gofman and Lindgren, 1950). Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.展开更多
In mammalians, the state of a somatic cell can be reversed from the terminal state to the totipotent state by means of somatic cell nuclear transfer (SCNT) (Gurdon, 1962) or induced pluripotent stem cells (iPSCs...In mammalians, the state of a somatic cell can be reversed from the terminal state to the totipotent state by means of somatic cell nuclear transfer (SCNT) (Gurdon, 1962) or induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). The DNA methylation and transcriptome profiles of embryonic stern cells (ESCs) derived from SCNT embryos (NT-ESCs) correspond closely to those of ESCs derived from in vitro fertilization embryos (IVF- ESCs). In contrast, iPSCs differ from both NT-ESCs and IVF-ESCs in that they retain the residual DNA methylation patterns of their parental somatic cells. As SCNT can be used to faithfully reprogram human somatic cells to pluripotency, it is ideal for cell replacement therapies (Ma et al., 2014). Following the successful production of the first human NT-ESCs (Tachibana et al., 2013) and the later gen- eration of human NT-ESCs based on cells from elderly adults or pa- tient cells (Chung et al., 2014; Yamada et al., 2014), a version of the SCNT technique for human therapeutics comes closer to reality. However, no matter what animal species or donor cell types are used in the cloned process, the cloning efficiency remains undesir- able. Besides, there are many phenotypic abnormalities in cloned animals, containing frequent embryonic and perinatal death and placentomegaly, and the underlying mechanisms remain unclear (Yang et al, 2007).展开更多
Objective To analyze the blastocyst formation and chromosome statuses of reconstructed embryos derived from human-goat interspecies somatic cell nuclear transfer (iSCNT), exploring the development retardant factors....Objective To analyze the blastocyst formation and chromosome statuses of reconstructed embryos derived from human-goat interspecies somatic cell nuclear transfer (iSCNT), exploring the development retardant factors. Methods Human specific point probes cep2, cep6, tel2 and 13q14.2, 21q22.13 combining fluorescence in-situ hybridization (FISH) technology were used to test trophectoderm cells of blastocyst and blastomeres of development arrest nuclear transfer (NT) embryos. Results A total of 209 reconstructed embryos were recovered, and the rate of blastocyst formation was 3.8% (8/209). FISH signals showed that chromosomal abnormalities were present in 2 blastocysts (2/8) and 146 development arrest embryos (146/201). Conclusion The rate of blastocyst formation is low, and reconstituted embryos of development arrest showed extensive chromosome abnormalities, suggesting that a chromosomal mechanism may underlie their developmental arrest.展开更多
The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned embryos.In this study we investigated the effect of DNA methylation state of donor cells on the d...The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned embryos.In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem(ES)cell nuclei.Our results confirmed that deletion of the DNA methyltransferase 3a(Dnmt3a)and DNA methyltransferase 3b(Dnmt3b)distinctly decreases the level of DNA methylation in ES cells.In contrast to wild type ES cells(J1),Dnmt3a–/–3b–/–(DKO)and Dnmt3b–/–(3bKO)donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer(ECNT)morula,blastocysts and postimplantation embryos(P<0.05).However,the efficiency of establish-ment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved,and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postim-plantation embryos was not altered either.Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.展开更多
Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the preve...Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.展开更多
The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine ...The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 mg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blas-tocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, re-spectively). The development rates to the blastocyst stage of NT embryos were significantly dif-ferent among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal ovi-duct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo develop-mental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.展开更多
Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) gen...Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.展开更多
Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as ...Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed em- bryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.展开更多
A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned em...A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.展开更多
Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic ...Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embry-onic ground state. Accumulating evidence indicates that in-complete or inappropriate epigenetic reprogramming of do-nor nuclei is likely to be the primary cause of failures in nu-clear transfer. This review summarizes the roles of various epigenetic mechanisms, including DNA methylation, histone acetylation, imprinting, X-chromosome inactivation, te-lomere maintenance and expressions of development-related genes on somatic nuclear transfer.展开更多
基金Key Research and Development Project of Hainan Province(ZDYF2021XDNY174)Science and Technology Major Project of Inner Mongolia(2021ZD0023–1)National Transgenic Key Project of the Ministry of Agriculture of China(2018ZX0800801B)。
文摘Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.
基金supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)
文摘The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout (KO) cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.
文摘Factors affecting the efficiency of nuclear transfer (NT) in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses (P〈 0.05), but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups (P〉0.05). When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos (P〈 0.05), although the fusion rate did not significantly differ among the three groups (P〉 0.05). Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage (P 〈 0.05), although the fusion rate was similar (P 〉 0.05). Activation of cytoplasts prior to fusion increased the cleavage rate (P〈 0.05) and blastocyst development (P〈 0.05) of reconstructed embryos, but decreased the fusion rate (P 〈 0.05) compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM + 3% OCS at the first 48 h and then cultured in TCM199+ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199+ 10% FCS or TCM199+3% OCS (P 〈 0.05). When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.
文摘The experiments of serial nuclear transfer were conducted between Boer goat and rabbit. The enucleated oocytes of rabbit were used as recipients while the blastomeres of goat morula was used as nuclear donor. The reconstructed embryos developing to morula were used as donor for serial cloning. As a result, two generations of reconstructed embryos were obtained, including 58 first generation reconstructed embryos and 14 second generation reconstructed embryos. The fusion rates were 79.5 and 70%, respectively, and there was no significant difference between them (P〉0.05). The cleavage rates were 75.9 and 28.6% respectively with significant difference (P〈0.01). No blastocyst was obtained from the second generation reconstructed embryos while 13.8% of first generation reconstructed embryos developed to blastocyst.
基金supported by grants from the Major State Basic Research Development Program of China(No.001CB5099)the National High Technology Research and Development Program of China(No.2001AA216121)+3 种基金National Natural Science Foundation of China(No.30040003)Projects of Shanghai Science&Technology Development Foundation(No.99DJ14002,00DJ14033,01DJ14003)the Chinese Academy of Sciences(No.KSCX-2-3-08)Shanghai Municipal Education Commission and by Shanghai Second Medical University
文摘To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.
基金Project (No. R-174-000-065-112/303) supported by the NationalUniversity of Singapore
文摘This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.
文摘Studies were carried out to find out the optimal parameters of enucleation and electroactivation ofrecipient rabbit oocyte for successful nuclear transfer,using the fluorescent stain,DAPI(4-6-diamidino-2-phenylindole),and electroactivation.According to the position of metaphasechromosomes in relation to the first polar body,the oocytes were classified into three types:1.thosewith chromosomes juxtaposed to the polar body;2.those with chromosomes in its adjacency; and3.those with chromosomes further removed.The relative proportions of each type appeared to varywith the time of maturation at which the oocytes were collected,with those of the later typesincreasing as the maturation process went further on.In addition,in-vitro cultivation ofelectroactivated oocytes gave the best results with oocytes that matured in-vito after injection ofovulating hormones(LH or HCG)and oocytes that were cultivated in-vitro for 17-19 hours.As aresult,it is recommended that oocytes be selected from those collected from the oviducts 13-15 hoursafter injection of LH or HCG,and electrofusion and electroactivation be done aftermicromanipulation and in-vito cultivation for 2-4 hours.By so doing,it is expected to achieve thehighest enucleation rate of oocytes and the highest fusion rate,the highest activation rate and thehighest development rate of the restructured embryos.
基金supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms,China(2014ZX08006-005 and 2014ZX0800950B)
文摘Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
文摘Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two(P<0.05).The development rate of embryos reconstruct-ed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization(FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that(1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA genotype through FISH.
文摘Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors (WHO). Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall (Gofman and Lindgren, 1950). Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.
基金supported by grants from the National Natural Science Foundation of China(No.31471395)to Q.Z.the Key Research Projects of the Frontier Science of the Chinese Academy of Sciences(QYZDY-SSW-SMC002)to Q.Z
文摘In mammalians, the state of a somatic cell can be reversed from the terminal state to the totipotent state by means of somatic cell nuclear transfer (SCNT) (Gurdon, 1962) or induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). The DNA methylation and transcriptome profiles of embryonic stern cells (ESCs) derived from SCNT embryos (NT-ESCs) correspond closely to those of ESCs derived from in vitro fertilization embryos (IVF- ESCs). In contrast, iPSCs differ from both NT-ESCs and IVF-ESCs in that they retain the residual DNA methylation patterns of their parental somatic cells. As SCNT can be used to faithfully reprogram human somatic cells to pluripotency, it is ideal for cell replacement therapies (Ma et al., 2014). Following the successful production of the first human NT-ESCs (Tachibana et al., 2013) and the later gen- eration of human NT-ESCs based on cells from elderly adults or pa- tient cells (Chung et al., 2014; Yamada et al., 2014), a version of the SCNT technique for human therapeutics comes closer to reality. However, no matter what animal species or donor cell types are used in the cloned process, the cloning efficiency remains undesir- able. Besides, there are many phenotypic abnormalities in cloned animals, containing frequent embryonic and perinatal death and placentomegaly, and the underlying mechanisms remain unclear (Yang et al, 2007).
基金supported by Grants from Special Fund for Excellent Young University teachers in Shanghai 2012Shanghai Science and Technology Developmental Foundations (Grant number: 09ZR1419000)
文摘Objective To analyze the blastocyst formation and chromosome statuses of reconstructed embryos derived from human-goat interspecies somatic cell nuclear transfer (iSCNT), exploring the development retardant factors. Methods Human specific point probes cep2, cep6, tel2 and 13q14.2, 21q22.13 combining fluorescence in-situ hybridization (FISH) technology were used to test trophectoderm cells of blastocyst and blastomeres of development arrest nuclear transfer (NT) embryos. Results A total of 209 reconstructed embryos were recovered, and the rate of blastocyst formation was 3.8% (8/209). FISH signals showed that chromosomal abnormalities were present in 2 blastocysts (2/8) and 146 development arrest embryos (146/201). Conclusion The rate of blastocyst formation is low, and reconstituted embryos of development arrest showed extensive chromosome abnormalities, suggesting that a chromosomal mechanism may underlie their developmental arrest.
基金supported by grants from the National Natural Science Foundation of China(Grant No.90919060)from the National Basic Research Program of China(No.2006CB944003).
文摘The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodel-ing of cloned embryos.In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem(ES)cell nuclei.Our results confirmed that deletion of the DNA methyltransferase 3a(Dnmt3a)and DNA methyltransferase 3b(Dnmt3b)distinctly decreases the level of DNA methylation in ES cells.In contrast to wild type ES cells(J1),Dnmt3a–/–3b–/–(DKO)and Dnmt3b–/–(3bKO)donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer(ECNT)morula,blastocysts and postimplantation embryos(P<0.05).However,the efficiency of establish-ment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved,and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postim-plantation embryos was not altered either.Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.
基金suppored by the Na-tional High-Tech Research and Development program (Grant No. 2006AA02Z113)
文摘Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.
文摘The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 mg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blas-tocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, re-spectively). The development rates to the blastocyst stage of NT embryos were significantly dif-ferent among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal ovi-duct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo develop-mental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.
文摘Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.
基金supported in part by the Major State Basic Research Development Program of China(Grant No.G20000161)State High-tech Research Development Program(Grant No.2003AA205102)the Natural Science Foundation of Beijing(Grant No.5030001).
文摘Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed em- bryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.
基金the National Natural Science Foundation of China (Grant Nos. 3030017630400232 and the Key International Cooperation Projects)
文摘A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.
文摘Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embry-onic ground state. Accumulating evidence indicates that in-complete or inappropriate epigenetic reprogramming of do-nor nuclei is likely to be the primary cause of failures in nu-clear transfer. This review summarizes the roles of various epigenetic mechanisms, including DNA methylation, histone acetylation, imprinting, X-chromosome inactivation, te-lomere maintenance and expressions of development-related genes on somatic nuclear transfer.