三种脂质体介导的花鲈胚胎干细胞转化效率的比较

Comparison of efficiency of three lipsomes mediated transformation to embryonic stem cells derived from Lateolabrax japonicus

通过3种脂质体(genejammer,genejuice和metafectene)介导绿色荧光蛋白基因转化花鲈胚胎干细胞,探讨不同脂质体介导的花鲈胚胎干细胞(LJES1)的转化效率,以及影响外源基因转化效率的因素,如细胞接种时间、初始接种密度、DNA和脂质体用量以及它们之间的比例等。实验发现,genejammer转化LJES1细胞效率最高,适合于该细胞系的转化。对于同一种脂质体,DNA与脂质体用量的比例对于绿色荧光蛋白基因转化LJES1最为重要,DNA与genejammer的比例为1∶6时,转化效率最高,为27.3%;DNA与genejuice的比例为1∶4时,转化效率最高,为12.1%;而DNA与metafectene的比例为1∶3时,转化效率达到最高,为5.3%。随着DNA、脂质体量的增加,转化效率有所提高,但达到一定的用量后,转化效率反而下降。

Sea perch embryonic stem cells （IAES1） are undifferentiated cell lines derived from mid-blastula of sea perch （ Lateolabrax japonicus）, and retained pluripotency in vitro, which provide a unique tool for introducing random or targeted genetic alterations. For improving the transformation efficiency of exogenous gene into LIES1 cells, a simple and effective method of gene transfer was established with several lipsomes transfection reagents, pCMV-EGFP plasmids were transformed by three lipsomes reagents （genejammer, genejuice and metafectene） into LIES1 cells. Expression of green fluorescent protein（GFP） in the cells was detected by fluorescence microscope equipped with mercury burner. For evaluating transformation efficiency, cells expressing GFP in 12-well plates were counted under fluorescent microscope. Transformation efficiencies of three lipsomes-mediated LIES1 cells were compared, and effects of various factors were examined, such as incubation time of cells, initial incubation density, the doses of DNA and lipsomes, and ratio of DNA and lipsomes, etc. It was demonstrated that the highest transformation efficiency was acquired by genejammer-rnediated GFP into LIES1, which was suitable for transformation of this cell line. For the same kind of lipsome, the ratio of DNA and lipsome for transformation of LJES1 is very important. It was found that 3 μL genejammer and 0.5 μg DNA gave rise to the optimal transformation efficiency of 27.3% for cells in 12 well plates with a ration of DNA and genejammer of 1：6; 4 μL genejuice and 1 μg DNA gave rise to the optimal transformation efficiency of 12.1% with a ration of DNA and genejuice of 1：4; and 6 μL metafectene and 2 μg DNA gave rise to the optimal transformation efficiency of 5.3 % with a ration of DNA and genejuice of 1 ： 3. The efficiency of transformation increases slightly as the increase of the DNA and lipsomes amount, but decreases after reaching a definite amount. Otherwise, initial incubation density and incubation time of cells affected the efficiency of transformation. 20×10^4 ·mL^-1 cells in 12 well plates are optimum, which should be 70% - 90% confluence. The higher efficiency of transformation arrived in 12 and 30 h after incubation, and cells should be rapid growth. ES cell-mediated gene transfer technique will be the most promising approach for producing site-mutated transgenic fishes with enhanced growth rate or disease-resistance and in analyzing functions of fish genes. Optimization of conditions for lipsome-mediated gene transfer with high efficiency would provide a technical foundation for genetic modifications in vitro.