Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring

Schürmann, A; Wells, David N.; Oback, Björn

doi:10.1530/rep-06-0054

Cited by 63 publications

(29 citation statements)

References 67 publications

(56 reference statements)

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“…In vitro production of embryos IVM oocytes were derived from Holstein-Friesian dairy cows as described (Schurmann et al 2006). IVM oocytes were fertilised at 20 to 22 h post-start of maturation as described previously (Thompson et al 2000).…”

Section: Incubation Of Sperm With Plasmid Dnamentioning

confidence: 99%

“…Then motile spermatozoa were collected after centrifugation at w1200 g for 20 min at room temperature and washed twice in TALP and then incubated with pEpi or pMax-GFP (1!10 6 spermatozoa with 1 mg plasmid in 60 ml of fertilisation medium). Fertilisation was performed in 50 ml IVF-SOF over a 24 h period as described (Schurmann et al 2006). For IVC, ten embryos were pooled in 10 ml ESOF medium and cultured for 7 days (D0: fertilisation).…”

Section: Incubation Of Sperm With Plasmid Dnamentioning

confidence: 99%

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Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

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Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (nZ96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (nZ751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68Z56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (nZ48) or GFP fluorescence (nZ94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

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Section: Incubation Of Sperm With Plasmid Dnamentioning

confidence: 99%

Section: Incubation Of Sperm With Plasmid Dnamentioning

confidence: 99%

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

View full text Add to dashboard Cite

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“…Recent studies, in which pronuclei were punctured before enucleation [27] or when enucleation was done before pronuclei formation [28], have proved that zygotes can also be successfully used as nuclear transfer recipients. In addition, Egli et al [7] showed that mouse zygotes temporarily arrested in mitosis could fully support somatic cell reprogramming.…”

Section: Discussionmentioning

confidence: 99%

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

et al. 2010

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Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

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“…This indicates the ooplasm of oocytes arrested at metaphase can support efficient nuclear reprogramming of transferred nuclei. More recently, early bovine zygotes were shown to support development of transferred somatic G1 phase nuclei when the maternal telophase II chromosomes and condensed sperm DNA were removed prior to pronuclei formation (Schurmann et al, 2006). In addition, the cytoplasm of enucleated prometaphase I (pro-MI) stage oocytes was found to be capable of remodeling ES cell nuclei, resulting in pseudo-pronuclei formation (Gao et al, 2002).…”

Section: Localization Of Nuclear Reprogramming Factorsmentioning

confidence: 99%

Nuclear reprogramming in zygotes

Lorthongpanich

Solter²,

Lim³

2010

Int. J. Dev. Biol.

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Nuclear reprogramming, the conversion of the epigenome of a differentiated cell to one that is similar to the undifferentiated embryonic state, can be facilitated by several methods, such as nuclear transfer, cell fusion, use of embryonic stem cell extracts, and more recently, by the introduction of exogenous transcription factors. Amongst these various strategies, somatic cell nuclear transfer (SCNT) is, by far, the most effective method of nuclear reprogramming. The majority of SCNT studies have been carried out using enucleated mature oocytes, as reprogramming is efficient and can be completed within hours following the introduction of the somatic cell nuclei into the recipient oocyte. Fertilized eggs, on the other hand, were regarded as poor recipients for nuclear transfer, as previous studies showed that embryonic blastomeres transferred into enucleated zygotes were unable to develop to blastocysts. However, more recent studies have demonstrated that the method of enucleation and the cell cycle phase of the embryos can impact the success of somatic cell reprogramming when zygotes were used as nuclear recipients. It is, therefore, timely to revisit and further explore the nuclear reprogramming capacity of zygotes as recipients for SCNT. Assessment of the various factors that influence the reprogramming capacity of zygotes in SCNT also provide hints of the mechanistic nature of nuclear reprogramming. KEY WORDS: nuclear transfer, reprogramming, zygote, embryo, SCNTDuring nuclear reprogramming, the cell-type specific epigenetic program of a differentiated cell is erased and the somatic genome re-acquires the potential to give rise to other cell types. In somatic cell nuclear transfer (SCNT), differentiated donor cells or nuclei are introduced into the cytoplasm of enucleated recipient cells to induce de-differentiation of the somatic genome to an embryonic state. Successful nuclear reprogramming will reset the transferred genome to totipotency. Thus, complete reprogramming of somatic genome via SCNT into embryos would result in normal embryonic development, giving rise to cloned animals that are genetically identical to the nuclear donor. The first mammal cloned from adult cell nuclei, Dolly the sheep, was successfully generated by SCNT in which nuclei of terminally differentiated mammary gland cells were transferred into enucleated mature oocytes and reprogrammed to a totipotent state (Wilmut et al., 1997). Since this breakthrough work, many other mammals such as mice (Wakayama et al., 1999), cattle (Kato et al., 1998), pigs (Polejaeva et al., 2000, cats (Shin et al., 2002), rabbits (Chesne et al., 2002;Li et al., 2006a), dogs (Lee et al., 2005, rats (Zhou et al., 2003), buffalos (Shi et al., 2007), ferrets (Li et al., 2006b), and camels (Wani et al., 2010) have been cloned by SCNT into Int. J. Dev. Biol. 54: 1631-1640 (2010) Abbreviations used in this paper: ECNT, embryonic cell nuclear transfer; ES, embryonic stem; GV, germinal vesicle; ICM, inner cell mass; MII, metaphase II; SCNT, somatic ce...

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Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring

Cited by 63 publications

References 67 publications

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Nuclear reprogramming in zygotes

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