Title: In vitro Culture of Bovine Fibroblast Cells and Human Lysozym Gene Transfection
Abstract: In order to offer doner cells for bovine transgenic cloning by nuclear transfering, bovine fibroblast cells were isolated by attaching tissue explants from ear skin of a bovine and purified by passage. The plasmid containing genes of human lysozym and two selecting marks(the EGFP and Neor genes) which express specifically in mammary gland was transfected into forth passages bovine fibroblast by Lipofectmine TM2000, and G418 selection (500 μg/mL) was applied since then. After 2 weeks, positive colonies were maintained in culture medium containing 250 μg/mL G418 for 2 ~ 3 passages. A small part of the positive cells were analyzed by PCR for gene integration, and karyotype was analyzed. Results showed that EGFP was expressed 24 h after transfection under fluorescence microscopy. The karyotype of the 15th passages was normal and diploid (60,XX). The fragment of 850 bp was detected by PCR, demonstrating that the foreign gene was integrated into the genome and could be expressed. The results indicate that the obtained cells may be competent for bovine transgenic cloning.
Publication Year: 2008
Publication Date: 2008-01-01
Language: en
Type: article
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