| China, as the primary country of pig-raising in the world, is abundant in resources of local inherited swine species. Nevertheless, due to the lag in swine breeding and weakness existing in feature exploring, utilization efficiency and protection toward local and introduced fine swine species, the fine genetic resources are not fully utilized, which considerably limits the level and benefit of swine production in China and makes the description of "primary" unbecoming. Consequently, it is one of essential tasks need to be solved for the sustainable development of swine breeding industry, that based on local inherited swine species and aided by modern biological technology such as somatic cell clone, transgene and embryotransfer, we should enrich the methods of resource protection and new species breeding, enhance the use ratio of local species, realize local species provision to cut down the amount of introduced species, and ultimately bring the dependence on foreign species introduction to the end. Additionally, researches concerning swine somatic cell clone and transgenic clone can not only significantly impel the advancement of stockbreeding, but also provide perfect animal models for human medicine and biology. However, some shortages of transgenic clone swine such as complicated procedure, high cost and low efficiency obstruct its direct benefiting human beings. Therefore, in an effort to employ cell culture technology to diversify the methods for protecting the resources of local inherited swine, provide sufficient somatic cell materials and establish the foundation for the production of transgenic clone swine, this research contains a series of experiments as followings:Experiment I, employing the ear skin of Anqing Liubai swine at the age of30days after birth and fetal tissues of Meishan swine at about40th day in pregnancy as the start materials, has separately cultivated fibroblasts cells methods of explant seeding culture or digestion by trypsin, and tested vitro biological characteristics of cell lines attained, which includes morphological observation of cell and depiction of cell growth curve. The results illustrate that the cell lines obtained from Anqing Liubai swine and Meishan fetal swine are in conformity with basic properties of typical fibroblast cells, i.e. the shape of the cells are spindle, star and polygon; cells join closely and grow in sheets; the growth curve display typical S-shaped. Using methods of explant seeding culture or digestion by trypsin, we successfully established19cell lines of4male Anqing Liubai Swine and15fetal Meishan Swine, which serve as plentiful materials for subsequent experiments.Experiment Ⅱ takes acquired cell lines of Meishan Swine(pig fetal fibroblasts cells, PFCs) as test materials, analyzes the conditions for transfection of Meishan Swine fibroblasts cells by liposome, and optimizes the conditions such as ratio of liposome to plasmid DNA, cell density, touching period of transfection kit, addition of serum and the implement of serum-free culture fluid. Finally, in6well plate where LipefectamineTM2000is adopted to transfect fibroblasts cell lines of Meishan Swine, the effects of transfected Meishan pig cells turns out to be optimal under the condition of cell density of80-90%,4μg of plasmid pEGFP-Nl,8μl of LipefectamineTM2000volume of sample, transfection without penicillin streptomycin combination and serum, and replacement by fresh, antibiotics-free culture medium after5h from transfection, with the transfecting efficiency of30%-40%approximately.Experiment III uses optimized system obtained from Experiment II to study transgenic clone processes of different Meishan Swine, to find high transfecting efficiency of fibroblasts cells of the No.6and No.13pigs. Transfection of two kinds of plasmids (pEGFP-Nl and DsRed) with LipefectamineTM2000reveals that the optimal volume of LipefectamineTM2000is3μg for both, and presents the phenomenon that some PFCs express both red and green fluorescent while others green fluorescent only, with no one merely expressing red fluorescent.Experiment IV conducts a tentative investigation on the conditions of electroporation transfecting method, in which BTX micropulser and Invitrogen neonTM micropulser are used to electronically transfect PFCs of Meishan pig. The survival rate of cells and the ratio of expressing green fluorescent2d after electrotransfection are selected as criterion for comprehensive evaluation. The results demonstrate that for BTX micropulser, the optimal values is170V/cm for elcetric field strength,15ms for pulse time and1pulse, while for Invitrogen neonTM micropulser,1400V/cm,30ms and1pluse.Experiment V applies PFCs of Meishan Swine which exhibit pEGFP-Nl obtained through lipofectin reagent,19acceptors in total, among which one gives birth to4clone piglets, with3piglets carrying EGFP gene.In summary, combining tissue culture, trypsin digestion and vitrification technology,this research establishes PFCs of Meishan Swine with tissues by trypsin digestion and tissues frozen and then recovery for the first time. The method of gene transfer by liposome that is suitable for PFCs of Meishan Swine is sieved out, with which transgenic clone swine are successfully produced. Positive Meishan pig cell lines are acquired via transferring two genes by liposome, which settles the foundation for producing clone pigs in the next step. Experiments also reveal that optimal parameters for electroporating PFCs of Meishan Swine varies with different brands of electroporation apparatuses, indicating parameters of a specific brand of electroporation apparatus need to be optimized and selected before electroporation transfecting method is applied in producing transgenic PFCs. |
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