Abstract
Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neor and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60–70 %) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0 %, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.
Similar content being viewed by others
References
Akers RM, Beal WE, McFadden TB, Capuco AV (1990) Morphometric analysis of involuting bovine mammary tissue after 21 or 42 days on non-suckling. J Anim Sci 68(11):3604–3613
Arat S, Rzucidlo SJ, Gibbons J, Miyoshi K, Stice SL (2001) Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes. Mol Reprod Dev 60(1):20–26. doi:10.1002/mrd.1057
Arat S, Gibbons J, Rzucidlo SJ, Respess DS, Tumlin M, Stice SL (2002) In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype. Biol Reprod 66(6):1768–1774
Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y (1999) Production of goats by somatic cell nuclear transfer. Nat Biotechnol 17(5):456–461. doi:10.1038/8632
Behboodi E, Ayres SL, Memili E, O’Coin M, Chen LH, Reggio BC, Landry AM, Gavin WG, Meade HM, Godke RA, Echelard Y (2005) Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer. Cloning Stem Cells 7(2):107–118. doi:10.1089/clo.2005.7.107
Bondioli K, Ramsoondar J, Williams B, Costa C, Fodor W (2001) Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Mol Reprod Dev 60(2):189–195. doi:10.1002/mrd.1076
Bordignon V, Keyston R, Lazaris A, Bilodeau AS, Pontes JH, Arnold D, Fecteau G, Keefer C, Smith LC (2003) Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells. Biol Reprod 68(6):2013–2023. doi:10.1095/biolreprod.102.010066
Brophy B, Smolenski G, Wheeler T, Wells D, L’Huillier P, Laible G (2003) Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein. Nat Biotechnol 21(2):157–162. doi:10.1038/nbt783
Chen SH, Vaught TD, Monahan JA, Boone J, Emslie E, Jobst PM, Lamborn AE, Schnieke A, Robertson L, Colman A, Dai Y, Polejaeva IA, Ayares DL (2002) Efficient production of transgenic cloned calves using preimplantation screening. Biol Reprod 67(5):1488–1492
Cho SK, Hwang KC, Choi YJ, Bui HT, Nguyen VT, Park C, Kim JH, Kim JH (2009) Production of transgenic pigs harboring the human erythropoietin (hEPO) gene using somatic cell nuclear transfer. J Reprod Dev 55(2):128–136
Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM (1998) Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280(5367):1256–1258
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):1–11
Duff K, Knight H, Refolo LM, Sanders S, Yu X, Picciano M, Malester B, Hutton M, Adamson J, Goedert M, Burki K, Davies P (2000) Characterization of pathology in transgenic mice over-expressing human genomic and cDNA tau transgenes. Neurobiol Dis 7(2):87–98. doi:10.1006/nbdi.1999.0279
Fujimura T, Murakami H, Kurome M, Takahagi Y, Shigehisa T, Nagashima H (2008) Effects of recloning on the efficiency of production of alpha 1,3-galactosyltransferase knockout pigs. J Reprod Dev 54(1):58–62
Gui T, Zhang M, Chen J, Zhang Y, Zhou N, Zhang Y, Tao J, Sui L, Li Y, Liu Y, Zhang X, Zhang Y (2012) In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos. Biotechnol Lett 34(8):1445–1452. doi:10.1007/s10529-012-0930-7
He S, Pant D, Schiffmacher A, Bischoff S, Melican D, Gavin W, Keefer C (2006) Developmental expression of pluripotency determining factors in caprine embryos: novel pattern of NANOG protein localization in the nucleolus. Mol Reprod Dev. 73, doi: 10.1002/mrd.20525
Heine WE, Klein PD, Reeds PJ (1991) The importance of alpha-lactalbumin in infant nutrition. J Nutr 121(3):277–283
Houdebine LM (2009) Production of pharmaceutical proteins by transgenic animals. Comp Immunol Microbiol Infect Dis 32(2):107–121. doi:10.1016/j.cimid.2007.11.005
Hyttinen JM, Korhonen VP, Hiltunen MO, Myohanen S, Janne J (1998) High-level expression of bovine beta-lactoglobulin gene in transgenic mice. J Biotechnol 61(3):191–198
Jang G, Bhuiyan MM, Jeon HY, Ko KH, Park HJ, Kim MK, Kim JJ, Kang SK, Lee BC, Hwang WS (2006) An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene. Theriogenology 65(9):1800–1812. doi:10.1016/j.theriogenology.2005.10.014
Keefer CL (2004) Production of bioproducts through the use of transgenic animal models. Anim Reprod Sci 82–83:5–12. doi:10.1016/j.anireprosci.2004.04.010
Khaliq S, Haider S, Ahmed SP, Perveen T, Haleem DJ (2006) Relationship of brain tryptophan and serotonin in improving cognitive performance in rats. Pak J Pharm Sci 19(1):11–15
Lee GS, Kim HS, Hyun SH, Lee SH, Jeon HY, Nam DH, Jeong YW, Kim S, Kim JH, Han JY, Ahn C, Kang SK, Lee BC, Hwang WS (2005) Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. Theriogenology 63(4):973–991. doi:10.1016/j.theriogenology.2004.04.017
McCreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE, Kind AJ (2000) Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405(6790):1066–1069. doi:10.1038/35016604
Meissner A, Jaenisch R (2006) Mammalian nuclear transfer. Dev Dyn 235(9):2460–2469. doi:10.1002/dvdy.20915
Neyroud N, Deschenes I, Akao M, Nuss HB, Marban E (2002) Somatic gene transfer of tagged K + channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes. J Membr Biol 190(2):133–144. doi:10.1007/s00232-002-1033-5
Ono Y, Shimozawa N, Ito M, Kono T (2001) Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol Reprod 64(1):44–50
Park KW, Lai L, Cheong HT, Cabot R, Sun QY, Wu G, Rucker EB, Durtschi D, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS (2002) Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts. Biol Reprod 66(4):1001–1005
Park JK, Lee YK, Lee P, Chung HJ, Kim S, Lee HG, Seo MK, Han JH, Park CG, Kim HT, Kim YK, Min KS, Kim JH, Lee HT, Chang WK (2006) Recombinant human erythropoietin produced in milk of transgenic pigs. J Biotechnol 122(3):362–371. doi:10.1016/j.jbiotec.2005.11.021
Rosen JM, Wyszomierski SL, Hadsell D (1999) Regulation of milk protein gene expression. Annu Rev Nutr 19:407–436. doi:10.1146/annurev.nutr.19.1.407
Rosenkrans CF Jr, Zeng GQ, MCNamara GT, Schoff PK, First NL (1993) Development of bovine embryos in vitro as affected by energy substrates. Biol Reprod 49(3):459–462
Sanchez-Munoz C, Sanz D, Zabala M (2007) Anthropometric characteristics, body composition and somatotype of elite junior tennis players. Br J Sports Med 41(11):793–799. doi:10.1136/bjsm.2007.037119
Svensson M, Hakansson A, Mossberg AK, Linse S, Svanborg C (2000) Conversion of alpha-lactalbumin to a protein inducing apoptosis. Proc Natl Acad Sci USA 97(8):4221–4226
Wang HL, Chang ZL, Li KL, Lian HY, Han D, Cui W, Jiao GZ, Wu YG, Luo MJ, Tan JH (2011) Caffeine can be used for oocyte enucleation. Cell Reprogram 13(3):225–232. doi:10.1089/cell.2010.0101
Wei J, Yang X, Zheng M, Wang M, Dai Y, Chen Z, Li N (2011) The recombinant chimeric antibody chHAb18 against hepatocellular carcinoma can be produced in milk of transgenic mice. Transgenic Res 20(2):321–330. doi:10.1007/s11248-010-9408-3
Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385(6619):810–813. doi:10.1038/385810a0
Zhao MT, Lin H, Liu FJ, Quan FS, Wang GH, Liu J, Hua S, Zhang Y (2009) Efficiency of human lactoferrin transgenic donor cell preparation for SCNT. Theriogenology 71(2):376–384. doi:10.1016/j.theriogenology.2008.08.004
Acknowledgments
This study was supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms (Project No. 2009ZX08008-009B) and Jiangsu Agriculture Science and Technology Innovation Fund (Project No. CX (11)4047).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Feng, X., Cao, S., Wang, H. et al. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer. Transgenic Res 24, 73–85 (2015). https://doi.org/10.1007/s11248-014-9818-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-014-9818-8