Skip to main content Accessibility help

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

  • Young-Hee Jeong (a1), Yeong Ji Kim (a1), Eun Young Kim (a2) (a3), Se Eun Kim (a1), Jiwoo Kim (a1), Min Jee Park (a2) (a3), Hong-Gu Lee (a4), Se Pill Park (a5) (a2) and Man-Jong Kang (a5) (a1)...


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.


Corresponding author

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:
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:


Hide All

These authors contributed equally to this work.



Hide All
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.
Bikfalvi, A., Klein, S., Pintucci, G. & Rifkin, D.B. (1997). Biological roles of fibroblast growth factor-2. Endocr. Rev. 18, 2645.
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.
Burgoyne, R.D. & Duncan, J.S. (1998). Secretion of milk proteins. J. Mammary Gland Biol. Neoplasia 3, 275–86.
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, 110.
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.
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.
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.
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, 111.
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.
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.
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.
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.
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.
Houdebine, L.M. (2000). Transgenic animal bioreactors. Transgenic Res. 9, 305–20.
Houdebine, L.M. (2009). Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 32, 107–21.
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.
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.
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.
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.
Laible, G. & Alonso-González, L. (2009). Gene targeting from laboratory to livestock: current status and emerging concepts. Biotechnol. J. 4, 1278–92.
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.
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.
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, 17
Porteus, M.H. & Carroll, D. (2005). Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–73.
Robl, J.M., Wang, Z., Kasinathan, P. & Kuroiwa, Y. (2006). Transgenic animal production and animal biotechnology. Theriogenology 67, 127–33.
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.
Sung, Y.H., Jin, Y., Kim, S.J. & Lee, H.W. (2014). Generation of knockout mice using engineered nucleases. Methods 69, 8593.
Szymczak, A.L. & Vignali, D.A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert. Opin. Biol. Ther. 5, 627–38
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.
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.
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.
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, 113.



Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed