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Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination

Published online by Cambridge University Press:  22 July 2015

Young-Hee Jeong ,
Yeong Ji Kim ,
Eun Young Kim ,
Se Eun Kim ,
Jiwoo Kim ,
Min Jee Park ,
Hong-Gu Lee ,
Se Pill Park  and
Man-Jong Kang
Young-Hee Jeong
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Yeong Ji Kim
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Eun Young Kim
Affiliation:
Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea. Mirae Cell Bio Research Institute, Mirae Cell Bio Inc., Seoul, Korea.
Se Eun Kim
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Jiwoo Kim
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Min Jee Park
Affiliation:
Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea. Mirae Cell Bio Research Institute, Mirae Cell Bio Inc., Seoul, Korea.
Hong-Gu Lee
Affiliation:
Department of Animal Biotechnology, KonKuk University, Seoul, Korea.
Se Pill Park*
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea, Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea.
Man-Jong Kang*
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea, Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
*
Se Pill Park. Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea. Tel: +82 64 754 4650. Fax: +82 2 6455 8759. E-mail: [email protected]
All correspondence to: Man-Jong Kang. Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea. Tel: +82 62 530 2113. Fax: +82 62 530 2129. e-mail: [email protected]
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Summary

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

Keywords

β-Casein geneCRISPR/Cas9 systemKnock-inTransgenic cattle
Type
Research Article
Information
Zygote , Volume 24 , Issue 3 , June 2016 , pp. 442 - 456
Copyright
Copyright © Cambridge University Press 2015 

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Footnotes

6

These authors contributed equally to this work.

References

Ahn, K.S., Kim, Y.J., Kim, M., Lee, B.H., Heo, S.Y., Kang, M.J., Kang, Y.K., Lee, J.W., Lee, K.K., Kim, J.H., Nho, W.G., Hwang, S.S., Woo, J.S., Park, J.K., Park, S.B. & Shim, H. (2011). Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts. Theriogenology 75, 933–9.CrossRefGoogle ScholarPubMed
Bikfalvi, A., Klein, S., Pintucci, G. & Rifkin, D.B. (1997). Biological roles of fibroblast growth factor-2. Endocr. Rev. 18, 26–45.Google Scholar
Bressan, F.F., Miranda, M.S., Bajgelman, M.C., Perecin, F., Mesquita, L.G., Fantinato-Neto, P., Merighe, G.F., Strauss, B.E. & Meirelles, F.V. (2013). Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer. In Vitro Cell Dev. Biol. Anim. 49, 250–9.CrossRefGoogle ScholarPubMed
Burgoyne, R.D. & Duncan, J.S. (1998). Secretion of milk proteins. J. Mammary Gland Biol. Neoplasia 3, 275–86.CrossRefGoogle ScholarPubMed
Cheng, X., Yu, X., Liu, Y., Deng, J., Ma, X. & Wang, H. (2013). Functional analysis of bovine Nramp1 and production of transgenic cloned embryos in vitro . Zygote 17, 1–10.Google Scholar
Cho, S.W., Kim, S.J., Kim, J.M. & Kim, J.S. (2013). Targeted genome engineering in human cells with the cas9 RNA-guided endonuclease. Nat. Biotechnol. 3, 230–3.Google Scholar
Clark, A.J. (1998). The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins. J. Mammary Gland Biol. Neoplasia 3, 337–50.CrossRefGoogle ScholarPubMed
Cui, X., Ji, D., Fisher, D.A., Wu, Y., Briner, D.M. & Weinstein, E.J. (2011). Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29, 64–7.Google Scholar
Denning, C. & Priddle, H. (2003). New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells. Reproduction 126, 1–11.CrossRefGoogle ScholarPubMed
Doppler, W., Groner, B. & Ball, R.K. (1989). Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line. Proc. Natl. Acad. Sci. USA. 86, 104–8.Google Scholar
Eigel, W.N., Butler, J.E., Ernstrom, C.A., Farrell, H.M. Jr., Harwalkar, V.R., Jenness, R. & Whitney, R. McL. (1984) Nomenclature of proteins of cow's milk: Fifth revision. J. Dairy Sci. 67, 1599–631.Google Scholar
Geurts, A.M., Cost, G.J., Freyvert, Y., Zeitler, B., Miller, J.C., Choi, V.M., Jenkins, S.S., Wood, A., Cui, X., Meng, X., Vincent, A., Lam, S., Michalkiewicz, M., Schilling, R., Foeckler, J., Kalloway, S., Weiler, H., Ménoret, S., Anegon, I., Davis, G.D., Zhang, L., Rebar, E.J., Gregory, P.D., Urnov, F.D., Jacob, H.J. & Buelow, R. (2009). Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433.Google Scholar
Hai, T., Teng, F., Guo, R., Li, W. & Zhou, Q. (2014). One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res. 24, 372–5.Google Scholar
Heintzman, N.D., Hon, G.C., Hawkins, R.D., Kheradpour, P., Stark, A., Harp, L.F., Ye, Z., Lee, L.K., Stuart, R.K., Ching, C.W., Ching, K.A., Antosiewicz-Bourget, J.E., Liu, H., Zhang, X., Green, R.D., Lobanenkov, V.V., Stewart, R., Thomson, J.A., Crawford, G.E., Kellis, M. & Ren, B. (2009). Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–12.CrossRefGoogle ScholarPubMed
Houdebine, L.M. (2000). Transgenic animal bioreactors. Transgenic Res. 9, 305–20.Google Scholar
Houdebine, L.M. (2009). Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 32, 107–21.Google Scholar
Kim, E.Y., Park, M.J., Park, H.Y., Noh, E.J., Noh, E.H., Park, K.S., Lee, J.B., Jeong, C.J., Riu, K.Z. & Park, S.P. (2012). Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the Oosight imaging system. Cell. Reprogram. 14, 305–11.CrossRefGoogle ScholarPubMed
Kubota, C., Yamakuchi, H., Todoroki, J., Mizoshita, K., Tabara, N., Barber, M. & Yang, X. (2000). Six cloned calves produced from adult fibroblast cells after long-term culture. Proc. Natl. Acad. Sci. USA. 97, 990–5.CrossRefGoogle ScholarPubMed
Kumar, S., Clarke, A.R., Hooper, M.L., Horne, D.S., Law, H.A.J., Leaver, J., Springbett, A., Stevenson, E. and Simons, P. (1994). Milk composition and lactation of β;-casein-deficient mice. Proc. Natl. Acad. Sci. USA. 91, 6138–42.Google Scholar
Kuroiwa, Y., Kasinathan, P., Matsushita, H., Sathiyaselan, J., Sullivan, E.J., Kakitani, M., Tomizuka, K., Ishida, I. & Robl, J.M. (2004). Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle. Nat. Genet. 36, 775–80.Google Scholar
Laible, G. & Alonso-González, L. (2009). Gene targeting from laboratory to livestock: current status and emerging concepts. Biotechnol. J. 4, 1278–92.CrossRefGoogle ScholarPubMed
McCreath, K.J., Howcroft, J., Campbell, K.H.S., Colman, A., Schnieke, A.E. & Kind, A.J. (2000). Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 29, 1066–9.Google Scholar
Mercier, J.C. & Gaye, P. (1980). Study of secretory lactoproteins: primary structures of the signals and enzymatic processing. Ann. N. Y. Acad. Sci. 343, 232–51.Google Scholar
Ni, W., Qiao, J., Hu, S., Zhao, X., Regouski, M., Yang, M., Polejaeva, I.A. & Chen, C. (2014). Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 9, 1–7 CrossRefGoogle ScholarPubMed
Porteus, M.H. & Carroll, D. (2005). Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–73.Google Scholar
Robl, J.M., Wang, Z., Kasinathan, P. & Kuroiwa, Y. (2006). Transgenic animal production and animal biotechnology. Theriogenology 67, 127–33.Google Scholar
Song, Y., He, X., Hua, S., Lan, J., Liu, Y., Cheng, P., Zhang, H., Li, J., He, X., Liu, J. & Zhang, Y. (2011). Construction of Ipr1 expression vector and development of cloned embryos in vitro . Zygote 21, 265–9.Google Scholar
Sung, Y.H., Jin, Y., Kim, S.J. & Lee, H.W. (2014). Generation of knockout mice using engineered nucleases. Methods 69, 85–93.Google Scholar
Szymczak, A.L. & Vignali, D.A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert. Opin. Biol. Ther. 5, 627–38Google Scholar
Tan, W., Carlson, D.F., Lancto, C.A., Garbe, J.R., Webster, D.A., Hackett, P.B. & Fahrenkrug, S.C. (2013). Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc. Natl. Acad. Sci. USA. 110, 16526–31.Google Scholar
Visel, A., Blow, M.J., Li, Z., Zhang, T., Akiyama, J.A., Holt, A., Plajzer-Frick, I., Shoukry, M., Wright, C., Chen, F., Afzal, V., Ren, B., Rubin, E.M. & Pennacchio, L.A. (2009). ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–8.Google Scholar
Wei, C., Liu, J., Yu, Z., Zhang, B., Gao, G. & Jiao, R. (2013). TALEN or Cas9 – rapid, efficient and specific choices for genome modifications. J. Genet. Genomics 40, 281–9.Google Scholar
Whitworth, K.M., Lee, K., Benne, J.A., Beaton, B.P., Spate, L.D., Murphy, S.L., Samuel, M.S., Mao, J., O'Gorman, C., Walters, E.M., Murphy, C.N., Driver, J., Mileham, A., McLaren, D., Wells, K.D. & Prather, R.S. (2014). Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol. Reprod. 91, 1–13.Google Scholar