ORIGINAL RESEARCH PAPER In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos Tao Gui • Meiling Zhang • Jianwen Chen • Yuanliang Zhang • Naru Zhou • Yu Zhang • Jia Tao • Liucai Sui • Yunsheng Li • Ya Liu • Xiaorong Zhang • Yunhai Zhang Received: 19 February 2012 / Accepted: 4 April 2012 / Published online: 24 April 2012 Ó Springer Science+Business Media B.V. 2012 Abstract A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were har- vested 24–72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombi- nant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1- hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats. Keywords Cancer cells Á Human lysozyme Á Mammary gland bioreactor Á Mouse mammary cancer cells Introduction Human lysozyme (hLYZ) forms part of the defensive machinery of organisms in preventing infection and tumors and immunological regulations and thus, has been widely used for medical and industrial purposes (Ibrahim et al. 2001). Natural hLYZ is extracted from human breast milk, neutrophils or urine. However, these sources are limited and the safety of the product cannot be guaranteed. Therefore, this approach is unsuitable for large-scale industrial production. Trans- genic animals as mammary bioreactors provide high levels of expression of an active product that can be purified without contamination, thus representing a novel method for large-scale production of recombi- nant proteins. However, this approach has yielded limited success to date. The choice of the genetic vector used for generation of transgenic animals requires preliminary verification of expression performance. Direct injection of mam- mary glands is a convenient approach in small animals, although it is associated with sampling problems and Electronic supplementary material The online version of this article (doi:10.1007/s10529-012-0930-7) contains supplementary material, which is available to authorized users. T. Gui Á M. Zhang Á J. Chen Á Y. Zhang Á N. Zhou Á Y. Zhang Á J. Tao Á L. Sui Á Y. Li Á Y. Liu Á X. Zhang Á Y. Zhang (&) Anhui Provincial Laboratory of Local Livestock and Poultry Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, Anhui, China e-mail: yunhaizhang@ahau.edu.cn 123 Biotechnol Lett (2012) 34:1445–1452 DOI 10.1007/s10529-012-0930-7