Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

Effect of synchronization of donor cells in early G1‐phase using shake‐off method on developmental potential of somatic cell nuclear transfer embryos in cattle

In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0‐phase (G0‐SCNT group) or early G1‐phase (eG1‐SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0‐phase and eG1‐phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake‐off method). The fusion rate in the G0‐SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1‐SCNT groups (39.2 ± 1.9%) (P < 0.05), but the developmental rates to the blastocyst stage of SCNT embryos per fused oocytes were similar for all groups. The overall production efficiency of the clone offspring in eG1‐SCNT groups (12.7%) per recipient cow was higher than that in G0‐SCNT groups (3%) (P < 0.05). The mean birth weight of cloned calves and the average calving score in the G0‐SCNT groups (48.1 ± 3.4 kg and 3.3 ± 0.3, respectively) was significantly higher (P < 0.05) than those of eG1‐SCNT groups (37.2 ± 2.1 kg and 2.3 ± 0.2, respectively). Results of this study indicate that synchronization of donor cells in eG1‐phase using the shake‐off method improved the overall production efficiency of the clone offspring per transferred embryo.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Assessment of reproduction and growth performance of offspring derived from somatic cell cloned pigs

Since cloned pig was successfully produced, a new opportunity for porcine breeding industry to conserve genetic resources has been opened. However, there has been no report to investigate whether both somatic cell nuclear transfer (SCNT) pigs and their offspring have the characteristics of the donor breed. In this study, we compared the reproductive and growth performance of American Large White boars cloned by SCNT with the donor boar, and analyzed the test parameters, including semen quality, re‐service rate, rate of parturition, and average daily gain. The results showed that these cloned boars and the donor boar had no significant differences in the tests (P > 0.05) and the growth performance of their offspring was similar to the naturally bred American Large White pigs. In summary, the reproductive and growth performance of cloned pigs are similar to the donor pig and within the normal range. This suggests that pigs cloned by SCNT have the potential to be used in reproduction and breeding.     

Treating Cloned Embryos,But Not Donor Cells,with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.     

哺乳动物体细胞核移植胚胎发育中表观重编程的研究进展

体细胞核移植（somatic cell nuclear transfer,SCNT）是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾（Xenopus laevis）幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。     

延边黄牛体细胞核移植的研究进展

牛体细胞核移植(somatic cell nuclear transfer,SCNT)是一套极其复杂的技术体系,它包括卵母细胞的成熟、供核细胞的准备、卵母细胞的显微操作、细胞融合、卵母细胞激活和胚胎培养。因此,许多因素影响着核移植胚胎发育。虽然陆续有克隆牛出生的报道,但是克隆效率依然低下。本综述对延边黄牛体细胞核移植体系的6个方面进行简单综述,以期为探究延边黄牛体细胞克隆的最优化条件、建立最优化的培养体系、提高克隆效率提供参考。     

Proper reprogramming of imprinted and non‐imprinted genes in cloned cattle gametogenesis

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non‐imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non‐cloned bulls. We found no differences between cloned and non‐cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha‐satellite and Art2) in oocytes recovered from cloned and non‐cloned cows. Again, no significant differences were observed between clones and non‐clones. These results suggested that imprinted and non‐imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring.     

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer

BackgroundSomatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.ObjectivesThis study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.MethodsThe viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer.ResultsMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption.ConclusionsSow urine-derived cells could be used to produce SCNT embryos.     

Research Advances on Somatic Cell Nuclear Transfer in Yanbian Yellow Cattle

Bovine somatic cell nuclear transfer (SCNT) is a sophisticated technique system,including oocyte maturation,donor cell preparation and oocytes microscopic operation,fusion,activation and culture.Although the birth of cloning cattle has been reported recently,the efficiency of somatic cell cloning has remained lowly.In order to establish the optimization somatic cell nuclear transfer system of Yanbian Yellow cattle,this review summarized only from 6 main aspects mentioned above in this field.     

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.     

The perspective of the incompatible of nucleus and mitochondria in interspecies somatic cell nuclear transfer for endangered species

Taking into account the latest Red List of the International Union for Conservation of Nature in which 25% of all mammals are threatened with extinction, somatic cell nuclear transfer (SCNT) could be a beneficial tool and holds a lot of potential for aiding the conservation of endangered, exotic or even extinct animal species if somatic cells of such animals are available. In the case of shortage and sparse amount of wild animal oocytes, interspecies somatic cell nuclear transfer (iSCNT), where the recipient ooplasm and donor nucleus are derived from different species, is the alternative SCNT technique. The successful application of iSCNT, resulting in the production of live offspring, was confirmed in several combination of closely related species. When nucleus donor cells and recipient oocytes have been used in many other combinations, very often with a very distant taxonomical relation iSCNT resulted only in the very early stages of cloned embryo development. Problems encountered during iSCNT related to mitochondrial DNA (mtDNA)/genomic DNA incompatibility, mtDNA heteroplasmy, embryonic genome activation of the donor nucleus by the recipient oocyte and availability of suitable foster mothers for iSCNT embryos. Implementing assisted reproductive technologies, including iSCNT, to conservation programmes also raises concerns that the production of genetically identical populations might cause problems with inbreeding. The article aims at presenting achievements, limitations and perspectives of iSCNT in maintaining animal biodiversity.     

Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm

Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear–mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8‐cell stage (P > 0.05). However, buffalo iSCNT embryos did not develop beyond the 16‐cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8‐ to 16‐cell stage (P > 0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P < 0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8–16‐cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear–mitochondrial interactions.     

Abnormal Expression of TIMP-2, SOD, Vimentin and PAI Proteins in Cloned Bovine Placentae

Cloned mammals suffer from high rates of placental abnormality and foetal loss during pregnancy. We previously used 2-D gel electrophoresis and mass spectrometry for global proteomic analysis of cloned and normal bovine placentae to identify differential protein expression patterns. Here, we used Western blot analysis to confirm the expression levels of several pregnancy-related proteins putatively identified as being differentially expressed in somatic cell nuclear transfer (SCNT) vs normal bovine placentae. The expression levels of tissue inhibitor of metalloproteinase-2 (TIMP-2), its downstream protein, matrix metalloproteinase-2 (MMP-2), superoxide dismutase (SOD), vimentin and plasminogen activator inhibitor-1 (PAI) were analysed in the placentae of SCNT cloned Korean native cattle that died immediately after birth and in normal placentae obtained by AI. Our results revealed that TIMP-2 and SOD were up-regulated in SCNT placenta compared with normal placenta, whereas MMP-2 levels were comparable in cloned and normal placentae, and vimentin and PAI were significantly down-regulated in SCNT compared with normal placentae. Our results suggest that key proteins of placental development are abnormally expressed in SCNT cloned bovine placentae, probably resulting in abnormal placental function and clonal mortality.     

Recent progress in bovine somatic cell nuclear transfer

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full‐term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT.     

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

To obtain the data concerning death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer (SCNT) and their progeny produced by Japanese institutions, a nationwide survey was carried out in July-August, 2006. As a result, lifetime data concerning 482 SCNT cattle (97.5% of cattle produced in the country at that time) and 202 progeny of SCNT cattle were accumulated and the death loss of these cattle was analyzed. Although 1/3 of delivered SCNT calves died during the perinatal period due to stillbirth and neonatal death, incidence of death loss due to diseases in SCNT cattle surviving more than 200 days after birth seems to be the same as these in conventionally bred cattle. In contrast, progeny of SCNT cattle showed the same level in death loss as observed in conventionally bred cattle throughout their lifetime. These results suggest that robust health would be expected in SCNT cattle surviving to adulthood and their progeny.     

Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system

Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% and 35.0%, respectively). Twenty-six SCNT embryos were transferred to 13 mares, and one mare delivered a live offspring at Day 342. There was a perfect identity match between the cloned foal and the cell donor after analysis of microsatellite DNA, and the mitochondrial DNA of the cloned foal was identical with that of the oocyte donor. These results demonstrated that the short disposable needle system can be used to recover oocytes to use as cytoplasts for SCNT, in the production of cloned foals and for other applications in equine embryology