Hostname: page-component-cd9895bd7-q99xh Total loading time: 0 Render date: 2024-12-27T06:26:30.806Z Has data issue: false hasContentIssue false
  • English
  • Français

Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre-loxP

Published online by Cambridge University Press:  25 January 2012

JoonHo Moon ,
SuJin Kim ,
HeeJung Park ,
JungTaek Kang ,
SolJi Park ,
OkJae Koo ,
Begona Roibas da Torre ,
Islam M. Saadeldin ,
ByeongChun Lee  and
Goo Jang
JoonHo Moon
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
SuJin Kim
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
HeeJung Park
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
JungTaek Kang
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
SolJi Park
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
OkJae Koo
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
Begona Roibas da Torre
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
Islam M. Saadeldin
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
ByeongChun Lee
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine and the Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
Goo Jang*
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea, 151-742.
*
All correspondence to: Goo Jang. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea, 151-742. Tel: +82 2 880 1280, Fax: +82 2 873 1269. e-mail: snujang@snu.ac.kr
Get access Rights & Permissions [Opens in a new window]

Summary

It is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with a plasmid that contained a red fluorescent protein marker (pCALNL-DsRed) and a floxed neomycin-resistance gene. Cells were selected with 750 μg/ml neomycin for 2 weeks following transfection but did not express DsRed after visualization under a fluorescence microscope. Expression was achieved only after transient transfection with plasmid DNA that expressed the Cre recombinase enzyme. The cells that expressed DsRed were used for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) were seen to have cleaved. Six blastocysts developed after SCNT and expressed DsRed. Deletion of the floxed neomycin-resistance gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in cloned blastocysts. This study demonstrated that Cre-loxP recombination can be conducted successfully in miniature pig fibroblasts and that the sequentially transformed cells can develop to the pre-implantation embryo stage via SCNT.

Keywords

Cre-loxPPorcine fibroblastSomatic cell nuclear transferTransfection
Type
Short Communication
Information
Zygote , Volume 20 , Issue 4 , November 2012 , pp. 423 - 425
Copyright
Copyright © Cambridge University Press 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ahn, C., Kim, J.Y., Lee, B.C., Kang, S.K., Lee, J.R. & Hwang, W.S. (2004). The past, present, and future of xenotransplantation. Yonsei Med. J. 45, 1017–24.CrossRefGoogle ScholarPubMed
Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F. & Stewart, A.F. (2009). Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–15.CrossRefGoogle Scholar
Branda, C.S. & Dymecki, S.M. (2004). Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28.CrossRefGoogle ScholarPubMed
Brault, V., Besson, V., Magnol, L., Duchon, A. & Herault, Y. (2007). Cre/loxP-mediated chromosome engineering of the mouse genome. Handb. Exp. Pharmacol. 29–48.CrossRefGoogle Scholar
Garcia-Escudero, V., Garcia-Gomez, A., Gargini, R., Martin-Bermejo, M.J., Langa, E., de Yebenes, J.G., Delicado, A., Avila, J., Moreno-Flores, M.T. & Lim, F. (2010). Prevention of senescence progression in reversibly immortalized human ensheathing glia permits their survival after deimmortalization. Mol. Ther. 18, 394–403.CrossRefGoogle ScholarPubMed
Koo, O.J., Jang, G., Kwon, D.K., Kang, J.T., Kwon, O.S., Park, H.J., Kang, S.K. & Lee, B.C. (2008). Electrical activation induces reactive oxygen species in porcine embryos. Theriogenology 70, 1111–8.CrossRefGoogle ScholarPubMed
Li, L., Pang, D., Chen, L., Wang, T., Nie, D., Yan, S. & Ouyang, H. (2009). Establishment of a transgenic pig fetal fibroblast reporter cell line for monitoring Cre recombinase activity. DNA Cell Biol. 28, 303–8.CrossRefGoogle ScholarPubMed
Nagy, A. (2000). Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109.3.0.CO;2-B>CrossRefGoogle ScholarPubMed
Nagy, A., Mar, L. & Watts, G. (2009). Creation and use of a Cre recombinase transgenic database. Methods Mol. Biol. 530, 365–78.CrossRefGoogle ScholarPubMed
Sauer, B. (1987). Functional expression of the Cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol. Cell Biol. 7, 2087–96.Google ScholarPubMed