Nuclear Transfer for Cloning Animals

Dinnyés, András; Tian, X.C.; Xu, Jie; Oback, Björn

doi:10.1002/3527600906.mcb.200400139

Cited by 2 publications

(3 citation statements)

References 138 publications

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“…This provides a long-sought molecular correlate for the elevated reprogrammability of quiescent donor cells into totipotency. Cell cycle co-ordination between the nuclear donor and MII-arrested cytoplast can increase cloning efficiency by 1) maintaining normal chromosome ploidy in the reconstructed NT embryo and 2) promoting epigenetic reprogramming of the donor genome [2]. In order to exclude ploidy-related reprogramming artefacts from G 2 /M [16] or S-phase nuclei [16,48], it was therefore important to ascertain that donors were synchronized in G 0 /G 1 .…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, differentiated cells rarely switch cell fates or produce daughter cells that do so. Radical manipulations, such as cloning by somatic cell NT (SCNT), force cells to lose their lineagespecific epigenetic marks and become totipotent again [2]. During NT, a donor nucleus is transplanted into an enucleated oocyte (cytoplast) where its epigenetic marks are cleared by ill-defined reprogramming factors.…”

Section: Introductionmentioning

confidence: 99%

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Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Kallingappa

Turner

Eichenlaub

et al. 2016

Biology of Reproduction

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Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Kallingappa

Turner

Eichenlaub

et al. 2016

Biology of Reproduction

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“…After transfer of the interphase nucleus, the MII environment with high levels of mitotic cyclin-dependent kinase (M-Cdk) complexes causes nuclear envelope breakdown (NEBD) and premature chromosome condensation (Lee and Campbell, 2006), while also reprogramming the donor epigenome to a totipotent state. After the diploid G 0 /G 1 donor cell nucleus has condensed from high M-Cdk activity, artificial activation (e.g., with the calcium ionophore ionomycin) decreases M-Cdk activity and initiates formation of a single pseudo-pronucleus (Dinnyes et al, 2016). This is dependent on subsequent treatment with protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), which ensures correct transition into interphase, while also preventing the extrusion of a pseudo-polar body (Szöllösi et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, andIn VivoSurvival of Cloned Sheep Embryos

McLean

Appleby

Fermin

et al. 2021

Cellular Reprogramming

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Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphasearrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

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Nuclear Transfer for Cloning Animals

Cited by 2 publications

References 138 publications

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, andIn VivoSurvival of Cloned Sheep Embryos

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