Study On Immortalization Of Chicken Embryo Fibroblasts

Fibroblasts are differeriated from mesenchymal cell during embryo period. They are the major cellular component of connective tissues. Because fibroblasts can be easily obtained , they have been widely used in many aspects of molecular and cell biology. Besides applied in the field of molecular and cell biology , chicken embryo fibroblasts have been used in the study of virology. They are also the main cellular resources for vaccine production. Like other normal somatic cells cultured in vitro, chicken embryo fibroblasts have limited lifespan. After a period of culture, chicken embryo fibroblasts will enter into a state of irreversible growth arrest and eventually die. We transfected primary chicken embryo fibroblasts with vector containing simian virus 40 LT antigen gene, so as to obtain such cells which can be cultured continuously in vitro.Primary chicken embryo fibroblasts were obtained by enzyme digestion culture method, using 9-10 days old chicken embryo. Primary chicken embryo fibroblasts were transfected with vector containing simian virus 40 LT antigen gene using lipofectin transfection method. The working concentration of puromycin for selection was determined at 1.25μg/mL. After selection with puromycin for 10-12 days, resistant cell colonies were obtained and expanded for continuous culture. Transfected cells remained unchanged significantly in morphology by far. Cells have been maintained in culture, up to 15 passages till now. The growth characteristics of cells remained unchaged apparently up to now. The cell growth curve indicated that the transformants have the typical growth character of "S". The transfected cells stayed in latent phase for 24-48 hours, then entered the logarithmic growth phase, during which cells grow rapidly. After the peak point at day 4-5 , cell number decreased gradually, which indicated that cells entered the stagnate phase. The predicted 558bp PCR product was obtained from the genome of transfected chicken embryo fibroblasts. A test for tumorigenicity of cells by soft agar cloning method was performed, there were no apparent clones observed with transfected chicken embryo fibroblast cells.Through continous passages and charaterization of transfected cells, the results indicated that the chicken embryo fibroblasts we obtained displayed potential capacity for continous passages in vitro.