Optimization Of Cell Culture And Transfection Conditions & Combined Effects Of Androgen And Estrogen On Mouse Uterus And Mammary Gland

Cell culture is an important and frequently-used technique in our research work and has been used in research field, including the localization of an interesting gene in cells, the expression of recombinant proteins, in vitro studies of the regulation of gene function, etc. Therefore conditions for cell culture should be optimized, such as the quality of water, serum and -mercaptoethanol , the kind of medium, and other factors . We ever prepared DMEM and MEM medium with Milli-Q water, double and six fold distilled water to culture mouse embryonic fibroblast cells. We found that the medium with 6 distilled water was the best of three methods, and the speed for cell multiplication in DMEM medium was faster than those in MEM. The quality of -mercaptoethanol is very essential. It is best to use the -mercaptoethanol special for cell culture. When manipulate on cells (e. g. prepare feeders for ES cells), we should use material that is harmful to cells as less as possible (e.g. using less Trypsin), manipulate as gently as possible (e.g. resuspending cells). One occurrence that every researcher who tries to grow mammalian cells in vitro has to deal with sooner or later is contamination. Sometimes it can be catastrophic. The best solution is to avoid contamination in the first place. If this is failing, contamination should be recognized as early as possible, the best way to get rid of contamination in cultures is to discard all contaminated cultures.To carry out further studies on gene expression regulationand mouse ES cell targeting, perfect cell transfection efficiency was acquired with the comparisons of three cell transfection assays (Electroporation, calcium phosphate method and lipofectin reagent). We found that the transfection efficiency of electroporation was linear with the voltage which was set from 350 to 450V; the transfection efficiency of calcium phosphate was greatly improved by using HEPES buffer; poor quality of DNA and longer incubating time were associated with decreased transfection efficiency of lipofectin. According to the characteristics of the three transfection protocols, transfection protocol can be optimized to obtain higher efficiency.