›› 2012, Vol. 32 ›› Issue (7): 955-.doi: 10.3969/j.issn.1674-8115.2012.07.029

• Technique and method • Previous Articles     Next Articles

Model establishment of transgenic mouse mammary epithelial cells for research of gene expression

JIANG Shi-zhong1,2, YAN Ya-bin1,2, XIE Fei1,2, WANG Juan1,2,3, HUANG Ying1,2,3, REN Zhao-rui1,2,3   

  1. 1.Shanghai Institute of Medical Genetics, Shanghai Children´s Hospital, Shanghai 200040, China； 2.Shanghai Institute of Medical Genetics, Shanghai Jiaotong University, Shanghai 200040, China； 3.Shanghai Key Laboratory of Embryo &|Production Engineering, Key Laboratory of Embryonic Molecular Biology, the Ministry of Health of China, Shanghai 200040, China
  • Online:2012-07-28 Published:2012-08-17
  • Supported by:

    National Science and Technology Major Program of China, 2009ZX08007-003B

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Abstract:

Objective To establish a cell model of transgenic mouse mammary epithelial cells for the research of foreign gene expression regulated by environment factors in the mammary gland as well as for the construction of mammary gland bioreactor. Methods The mammary epithelial cells of human transferrin (hTF) transgenic mouse were obtained by mechanical disruption and collagenase digestion, and followed by primary culture. After purification by trypsase, the growth curve of mammary epithelial cells was drafted. The expression of keratin 18 was detected by immunohistochemistry, the ultrastructure of mammary epithelial cells was observed by transmission electron microscopy, the distribution of cell cycles was determined by flow cytometry, and the karyotype of mammary epithelial cells was analysed under microscope. Eukaryotic expression vectors of bovine prolactin (pCMV-bPRL) were transfected into transgenic mammary epithelial cells, and the expression of exogenous bPRL was detected. Results The growth curve of mammary epithelial cells exhibited “S” shape. Immunofluorescence analysis revealed that there was positive expression of keratin 18 in hTF transgenic mouse mammary epithelial cells. Transmission electron microscopy demonstrated that the nuclei were bigger in mammary epithelial cells, with abundant cytoplasm, vacuole, rough endoplasmic reticulum and golgi body. Flow cytometry indicated that the proliferation of mammary epithelial cells was active, and cells in G2/M phase or S phase accounted for 15%. Cells in division stages had normal diploid and intact chromosome. Twenty-four hours after transfection of mammary epithelial cells by pCMV-bPRL, the expression of bPRL was detected in the supernatant. Conclusion The transgenic mouse mammary epithelial cells cultured in vitro have in vivo biological characteristics and capacity of expression of eukaryotic gene vectors, which may provide a good cell model for investigation of environment factors on the function of mammary gland and generation of mammary gland bioreactor.

Key words: transgenic mouse, mammary epithelial cell, eukaryotic gene vector, bovine prolactin