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Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved., (© 2021. The Author(s).)

This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1-60 and TRA-1-81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: beta-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis (BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P<0.05), number of inner cell mass cells (13.3+/-4.3 vs. 7.6+/-2.2, P<0.05), and total cells (57.9+/-19.5 vs. 36.3+/-8.2, P<0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0mM calcium in fusion/activation medium (1.0C), and 7.5mug/mL cytochalasin B treatment (0.1C&CB) yielded higher rates (P<0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased (P<0.05) in 1.0C (77.4+/-28.9) compared to the control (58.5+/-22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1C&CB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes (BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts., (Copyright 2005 Wiley-Liss, Inc)

It was recently shown that treatment of cloned embryos with histone deacetylase inhibitors improves efficiency for the success rate of developmental potential to term in several species. The objective of the present study was to investigate the influence of the histone deacetylase inhibitor Scriptaid (Sc) on in vitrodevelopment in early porcine SCNT embryos and on their gene expression pattern. Based on the findings of previous porcine studies (Zhao et al.2009), the reconstructed oocytes were treated with 500nM Scriptaid for 14 to 16h after post-fusion activation (6-DMAP/demecolcine). In our preliminary study, blastocyst rate significantly increased in the Sc-treated group, compared with the control group (25.1±2.8% and 13.8±1.9%, respectively, P<0.05). We determined gene expression using quantitative real-time RT-PCR. The results showed that OCT3/4 gene was expressed at a similar level in in vivoand SCNT blastocysts with/without Sc. IGF2 and H19 genes tended to be highly expressed in both SCNT blastocysts with (1.6-fold and 3.1-fold, respectively) and without (2.0-fold and 5.8-fold, respectively) Sc than that of the in vivoblastocysts. We found differences in imprinted gene expression patterns between in vivoand cloned blastocysts. Expression of H19 and IGF2 in SCNT blastocysts after Scriptaid treatment decreased towards the expression levels of in vivoblastocysts. These results indicated that Scriptaid treatment in SCNT embryos may also have beneficial effects on in vitrodevelopmental competence as well as their gene expression pattern. [ABSTRACT FROM AUTHOR]

Bovine transgenic embryonic stem (ES) cells have not been reported yet because it seems that the derivation methods and the culture conditions for the inner cell mass are neither consistent nor optimized. Isolation of inner cell mass and primary culture of ES colonies is a critical step toward the establishment of authentic bovine ES cell lines. Herein, we reconstructed somatic cell nuclear transferred (SCNT) bovine blastocysts carrying a vector expressing the human INF-αgene, and isolated inner cell masses to derive transgenic bovine embryonic stem cells. In addition, we added 2 inhibitors, inhibition (2i system) of the mitogen-activated protein kinase (Erk1/2) cascade, PD0325901(3 Î1/4M), and of glycogen synthase kinase 3, CHIR99021 (1 Î1/4M), in the inner cell mass primary culture to check reliability of the 2i system for bovine ES culture. The 2 inhibitors made the morphology of colonies more intact, and primary colonies were better maintained in early passages. However, there were no significant effects on the attachment rate and maintenance in late passages (percent of percent over 3 passages: 2i system, 21/38 (55.3%); control, 22/42 (33.3%); P<0.05). Inner cell masses were isolated mechanically and subcultured by an enzymatic in primary inner cell mass culture. Massive growth of trophoblast cells appears to inhibit inner cell mass growth, so hatching and hatched blastocysts were cut with a needle to remove trophoblast cells. Poor quality blastocysts were attached by the whole seeding method, and the margin trophoblast cells were consecutively removed in early passages. Established bovine ES cells express alkaline phosphatase, Oct-4, SSEA1, SSEA4, Tra-1–60, and Tra-1–81. We confirmed pluripotent gene expression of bovine ES like cells; Oct-4, SSEA1, and Rex 1were positive, but trophoblast marker CDX2was negative. This study shows that the 2i system is a reasonable method for use during inner cell mass culture in early passages. We established 6 transgenic nuclear transfer bovine ES cell lines with the 2i system and 4 in vitrofertilized bovine ES cell lines (all were over 10 passages). [ABSTRACT FROM AUTHOR]

In bovine somatic cell nuclear transfer (SCNT), oocyte activation is an essential element for the successful development of cloned embryos. Chemical treatment with ionomycin induces transient levels of Ca2+, and this has been used in the activation of reconstructed embryos. In vitro-fertilized oocytes are naturally activated by sperm following several Ca2+transients known collectively as Ca2+oscillations. We aimed to improve the developmental efficiency of embryos produced by SCNT by mimicking this Ca2+oscillation artificially. SCNT was performed as follows; bovine calf skin fibroblast cells were transferred into the perivitelline space of IVM oocytes, and then these NT couplets were treated with electrical fusion (2 pulses, 1.75 kV cm-1, 15 μs). Reconstructed embryos were subsequently cultured in SOF medium (5% CO2, 5% O2, and 38°C). To mimic Ca2+oscillation, we carried out 3 different repetitive ionomycin (10 μM) treatments at 1-h intervals. There were 3 groups: group 1 (4 min, 1 time), group 2 (30 s, 4 times), and group 3 (1 min, 4 times). The difference in embryo development amongst these experimental groups was then analyzed using the one-way ANOVA test after arcsine transformation to maintain homogeneity of variance. All analyses were performed using SAS (version 8.1, SAS Institute, Cary, NC, USA). Significant differences among the treatments were determined when the P-value was <0.05. In experiment 1, to assess developmental efficiency, the cleavage rate was investigated on Day 2 and the formation rate of blastocysts (BL) was examined on Day 7. In group 3, a significant increase in BL formation was observed [47/263 (17.8%), 50/259 (19.3%), and 67/258 (26.0%), respectively]. In experiment 2, culturing each group of embryos with different ionomycin treatments caused no significant differences among the groups in terms of the total number of BL (164.3, 158.5, and 145.1, respectively). In experiment 3, expression of apoptosis-related genes in each group was evaluated by real-time PCR and the TUNEL assay. The 3 BL within each group were analyzed for the expression of apotosis-related genes. Expression of the anti-apoptotic Bcl-2 gene was increased in group 3, whereas the expression of the pro-apoptotic Bax was decreased. A decrease in the number of apoptotic nuclei was also observed in group 3. In conclusion, the present study demonstrated that repetitive ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis. [ABSTRACT FROM AUTHOR]