Piglets produced from cloned blastocysts cultured in vitro with GM‐CSF

Lee, Kiho; Redel, Bethany K.; Spate, Lee D.; Teson, J.; Brown, Ashley N.; Park, Kwang-Wook; Walters, Eric M.; Samuel, Melissa; Murphy, Clifton N.; Prather, Randall S.

doi:10.1002/mrd.22143

Cited by 65 publications

(37 citation statements)

References 43 publications

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“…In this study, the overall frequency of blastocysts from conventional activation or SCNT tended to be lower compared to our previous report [38]. This could be seasonal observation; however, we believe this is due to the difference in the source of oocytes.…”

Section: Discussioncontrasting

confidence: 63%

Pig oocyte activation using a Zn2+ chelator, TPEN

et al. 2015

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Artificial oocyte activation is a critical step during somatic cell nuclear transfer (SCNT). Most of current activation protocols focus on inducing an increase in the intracellular free Ca2+ concentration of the oocyte. Here, we have utilized a zinc chelator, TPEN, to enhance the efficiency of oocyte activation during SCNT. TPEN treatment of matured pig oocytes resulted in the reduction of available Zn2+ in pig oocytes; however, the cytosolic Ca2+ concentration in the oocytes was not affected by the TPEN treatment. When various concentrations (100 – 250 μM) and incubation durations (45 min – 2.5 hours) of TPEN were used to activate oocytes, the efficiency of oocyte activation was not different from conventional activation methods. When oocytes that were activated by conventional activation methods were incubated with a lower concentration of TPEN (5 – 10 μM), a significant increase in embryos developing to the blastocyst stage was observed. In addition, when oocytes receiving a small Ca2+ stimulus were further activated by higher concentration of TPEN (100 – 200 μM), a significant increase in the frequency of blastocyst formation was observed, compared to a conventional activation method. This result indicated that TPEN can be a main reagent in oocyte activation. No increase in the cytosolic Ca2+ level was detected when oocytes were exposed to various concentrations of TPEN, indicating the ability of TPEN to induce oocyte activation is independent of an intracellular Ca2+ increase. We were able to produce clones through SCNT using the TPEN-assisted activation procedure, and the piglets produced through the process did not show any signs of abnormality. In this study, we have developed an efficient way to utilize TPEN to increase the developmental potential of cloned embryos.

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Section: Discussioncontrasting

confidence: 63%

Pig oocyte activation using a Zn2+ chelator, TPEN

et al. 2015

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“…In pigs, modified Tris-buffered medium (mTBM) is known to be proper to minimize polyspermy not affecting to the penetration rate [13,14]. For successful IVF, viability of sperm needs to be monitored prior to the IVF.…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-Mediated Gene Targeting during Embryogenesis in Swine

Ryu

Lee

2017

Methods in Molecular Biology

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Ability to disrupt genes is essential in elucidating gene function. Unlike rodents or amphibians, it has been difficult to generate gene targeted embryos in large animals. Therefore, studies of early embryo development have been hampered due to the limitation. A recent technology suggests that targeted mutations can be successfully introduced during embryogenesis, thus by-passing the need of breeding to produce gene-targeted embryos. This is particularly important in large animal models because of longer gestation period and higher animal cost. Here we describe a specific approach to disrupt up to two genes simultaneously during embryogenesis using the CRISPR/Cas9 technology in swine. The approach can help understand the mechanism of zygotic genome activation in large animals.

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“…Injected zygotes were washed then transferred and cultured into PZM-3. Embryos used for embryo transfer were cultured in PZM-3 in the presence of 10 ng/ml GM-CSF [15] until embryo transfer. A total of 245 microinjected embryos were transferred into two surrogate sows at day 5 or 6 post-IVF.…”

Section: Methodsmentioning

confidence: 99%

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs

Kang¹,

Cho²,

Ryu

et al. 2016

Reprod Biol Endocrinol

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AbstractsBackgroundPigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype.MethodsFirst, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer.ResultsThree live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen.ConclusionsHere, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0206-5) contains supplementary material, which is available to authorized users.

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Piglets produced from cloned blastocysts cultured in vitro with GM‐CSF

Cited by 65 publications

References 43 publications

Pig oocyte activation using a Zn2+ chelator, TPEN

Pig oocyte activation using a Zn2+ chelator, TPEN

CRISPR/Cas9-Mediated Gene Targeting during Embryogenesis in Swine

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs

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