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Studies on Construction of Specific Gland Expressional Vector of HLF and Modified Fibroblast Cell Line

Author: MengLi
Tutor: WangFeng
School: Nanjing Agricultural College
Course: Animal Genetic Breeding and Reproduction
Keywords: mammary gland expressional vector random insertion site-specific integration expression in vivo modified cells line
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 20
Quote: 0
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Abstract


Human lactoferrin (hLF) is a multifunctional glycoprotein of 80 kD secreted in many tissue fluids including tears, saliva, semen, vaginal secretion, milk, and plasma. Both in vitro and in vivo evidence indicate that hLF is involved in iron absorption in the intestinal tract as well as in broad-spectrum primary defense against bacteria, fungi, protozoa and viruses. In addition, several studies also suggest that hLF modulates the inflammatory response, regulates gene expression, and promotes bone growth. These bioactivities suggest that hLF may have important therapeutic applications, such as in proPHylaxis treatment, nutritional supplementation, and food and or medicine preservation. Therefore, market demand for hLF is primed to expand dramatically. A number of attempts have been made to produce recombinant human lactoferrin (rhLF) using prokaryotic and eukaryotic expression systems. However, problems such as low protein expression level, lack of accurate post-translational modifications as well as complex purification procedures have made current approaches unsuitable for large-scale production. As such, a goat mammary bioreactor would be an excellent system for large-scale production of rhLF because of its established faithful incorporation of post-translational modifications and efficiency for purification of heterogonous proteins.So we construct a mammary gland-specific expressional vector pGBC-hLF, a random inserted vector. In order to testify the expression of it in vivo, Neo gene was inserted into pGBC-hLF and was transfected into the goat mammary gland epithelium cells GMC and mouse mammary tumor epithelium cells line C127, which laid a experimental foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells. So as to prepare the hLF gene transgenic goat fetal-derived fibroblast cells, the double marker genes of Neo and EGFP were sub-cloned into the plasmid vector pGBC-hLF and was transfected into the goat fetal-derived fibroblast cells cultivated in the conditional medium, which can shorten the time for selection by G418 and facilitate the selection of modified cells by EGFP gene. For achieving the goal of getting the sit-specific integration mammary bioreactor for hLF cDNA and eliminate the impact of position effect in the random integration modified animals, we constructed the targeting vector at the lotus in (3-casein gene belonging to milk protein genes, and further we analyzed the bioactivity of the gene-targeting vector, which offer some basis for the production of gene-targeted cells in the future.The concrete content of our experiment are as follows:(1) The aim of this study was to construct a mammary gland-specific expressional vector pGBC-hLF-NEO for Human Lactoferrin(hLF) Gene and testing its expression in the mammary gland epithelium cells. The constructed vector contains the 6.2 kb 5’flank regulation region sequence including promoter,extronl, intron 1 and part of extron 2 of goatβ-casein gene and 7.1 kb 3’flank regulation region sequence including transcriptional ending signal of goatβ-casein gene with hLF gene locating between them. A cassette of Neo gene was also inserted into the vector and the total length of the vector is 26.736kb. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, the lined vector DNA by Sal I was transfected into the dairy goat mammary gland epithelium cells and C127 cells by LipofectamineTM 2000. 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 the transfected GMC cells and C127 cells individually. After proliferation culture, The two kinds of transgenic cells were cultured in induction medium containing serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone, which could induce recombinant hLF expression. RT-PCR and Western-blotting analysis showed that the constructed mammary gland specific vector pGBC-hLF-Neo has the whole bioactivity to efficiently express hLF in both mammary gland cells and secrete the protein into outside of the cells. At the same time, it was first time to confirm that the mouse mammary tumor epithelium cells line C127 equally valid to normal goat mammary epithelium cells in testing the bioac tivity of mammary gland specific expressional vector. Above all, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.(2) we constructed a vector pGBC-hLF-Neo-IRES2-EGFP harboring double markers of Neo and EGFP and verify the identification o f this vector by enzyme digestion, PCR, and sequencing. The goat fetal-derived fibroblast cells were transfected by this vector and had been selected for 7-10 d by G418, then we get the cell clones expressing the green fluorescent protein. We used 96-well cell culture plates to isolate cell lineages obtained from a single or several fibroblast cells expressing the green fluorescent protein. Since little mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. Which is more favorable for the formation of cell clone expressing the green fluorescent protein? Reliable hLF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence and this can effectively avoid the problem of false positive problem lead by single-gene PCR. This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant PHarmaceuticals from the milk of transgenic animals.(3) we constructed the targeting vector at the lotus inβ-casein gene belonging to milk protein genes, The 5’homologous arm was 6.3 kb fragment including promoter, exon 1, intron 1 and part of exon 2 of the goat beta-casein gene sequence, and the 3’homologous arm was 2.4 kb long fragment including the exon 8, intron 8 and exon 9. The Neo gene, positive selection marker gene, was located between two loxp sites, single directional repeated sequence. The HSV-tk gene, was located outside the homologous recombinant area as negative selection marker gene. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyse the bioactivity of the vector, linearised Pgbc5-hLF was transfected into goat mammary epithelial cell GMC by using lipofectamine. After selection with G418 for 8-10 days, G418-resistant cell clones were obtained. PCR analysis demonstrated that hLF cassette had been integrated into the genomic DNA of the transfected cells. After prolife ration culture, the transgenic cells were cultured in induction medium containing serum-free medium with prolactin, insulin and hydrocortisone which can induce recombinant human hLF expression. RT PCR and Western-blotting analysis showed that the induced cells produced recombinant human hLF mRNA and protein. The results show that the constructed targeting vector has bioactivity to efficiently express hLF in mammary gland cells and secrete the protein outsite of the cells.

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