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Improvement of Cloning Efficiency in Minipigs Using Post-thawed Donor Cells Treated with Roscovitine

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Abstract

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P < 0.05) and without roscovitine (78.6 % ±5.5) (P < 0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P < 0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6 months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54 %. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1 h prior to nuclear transfer.

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Acknowledgments

This study received Grant support from the Agenda Program (No. PJ008587) and (PJ009095), Rural Development Administration (http://www.rda.go.kr), Republic of Korea. The authors appreciate Professor Hochi Shinichi (Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan) for his helpful discussions to improve this article.

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Authors and Affiliations

  1. Animal Biotechnology Division, National Institute of Animal Science, RDA, Gyeonggi-do, 441-706, Republic of Korea

    Seongsoo Hwang, Keon Bong Oh, Dae-Jin Kwon, Sun-A Ock, Gi-Sun Im, Sung-Soo Lee & Jin-Ki Park

  2. Development and Differentiation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Republic of Korea

    Jeong-Woong Lee

  3. Department of Animal Sciences, The Ohio State University, 2029 Fyffe Rd, Columbus, OH, 43210, USA

    Kichoon Lee

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  1. Seongsoo Hwang
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  2. Keon Bong Oh
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  3. Dae-Jin Kwon
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  4. Sun-A Ock
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  5. Jeong-Woong Lee
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  9. Jin-Ki Park
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Correspondence to Seongsoo Hwang or Jin-Ki Park.

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Hwang, S., Oh, K.B., Kwon, DJ. et al. Improvement of Cloning Efficiency in Minipigs Using Post-thawed Donor Cells Treated with Roscovitine. Mol Biotechnol 55, 212–216 (2013). https://doi.org/10.1007/s12033-013-9671-7

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