Construction and identification of mammary expressional vector for cDNA of human lactoferrin / 生物工程学报

The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5' flank regulation region including promoter, other elements and the 7.1 kb long 3' flank regulation region including transcriptional ending signal of a goat's beta-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat's mammary gland epithelium cells and C127 cells of a mouse's mammary epithelium by Lipofectamine. After selection with G418 for 8-10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.

Identification methods of embryonic stem cells / 中国组织工程研究

OBJECTIVE: Embryonic Stem Cell (ES) are characterized by totipotency and normal karyotype and provide the theoretical base in the following fields: embryonic developmeut of mammalian, cell differentiation, expres sion of exogenous genes and utilization of Ess to develop animal model for human inherited diseases. The identification of ES therefore is very important to research and utilize ES.DATA SOURCES: The relevant articles to embryonic stem cell between January 1980 and December 2003 were computer searched for in Medline with the key words "embryonic stem cell, embryo, Alkaline phosphatase,Oct-4"in English. Similarly, the relevant articles to embryonic stem cell between January 1980 and December 2003 were computer searched for in China Journal Full-text Database (CJFD) with the key word "embryonic stem cell" in Chinese.STUDY SELECTION: The articles were browsed firstly. The inclusion criteria were for those articles about the identification of embryonic stem cells. The exclusion criteria were for those about repetitive studies, reviews and other similar articles.DATA EXTRACTION: 16 articles about the identification of embryonic stem cells were collected. Then, the full-texts of the articles were looked through.DATA SYNTHESIS: The selected data were summarized in the following order: ①Preliminary identification of Ess based on morphology and growth;②Immunological valuation; ③Chromosome related identification; ④Identi fication of totipotency and pluripotency.CONCLUSION: The identification of embryonic stem cells is not the result of only one identifying method, but a process of identification. During this process of comprehensive identification, it is recommend to conduct AKP test firstly, karyotype analysis secondly, then examination of surface markers and finally identification of Ess totipotency when Ess are sufficient, takingcare to repeat every identification.