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Huh-7-luc肝癌细胞

1.       细胞名称    Huh-7-luc

2.       组织  肝癌

3.       母细胞来源  ATCC

4.      转染方法与标记过程的描述：慢病毒转染筛选单克隆 

Construct Luciferase expressing vector pWPXL-Luc.

pGL3-control was first digested with XbaI and then blunted with Klenow.The resulted DNA was then digested with BglII. The DNA fragment (1902 bp)encoding luciferase was then ligated to BamHI/SmaI backbone of pWPXL.

Preparation of the lentivirus.

In the first day, 2.5×10⁶ of 293Tcells was plated in 250px plate.

In the next day, 20μg of pWXL-Luc, 15μg of psPAX2 and 6μg of PMD2G was diluted into a tube containing 500 μl H₂O，and then 500μl 2xHBS wassupplemented into the tube。50μl of 2.5M CaCl2 was added and mixed gently. The solution wasincubated 20~25min at RT。After that, thesolution was added into the medium。After 6 hr, the medium was replaced withfresh medium。

The fourth day, medium was collected at 3000 rpm, RT for 5 min. Thevirus was purified by passed through with 0.45 μm membranefilters.

Establishment of HuH-7-Luc cell line.

pWXL-Luc Virus were used to infect the huh-7 cells at MOI=1000 in the presence of 4μg/ml polybrene. After 3 days, cells were counted, diluted and plated into96-well plate by about one cell per well. After 2~3 weeks, the expression ofthe luciferase in the cell clones was examined. Cell clone with high expressionof luciferase was selected and stored.

5.      培养条件：置于37℃、5％CO₂培养箱中，用含10％胎牛血清（FBS;    GIBCO）和1％双抗（100 U/ ml青霉素和100 μg/ ml链霉素，GIBCO）的DMEM（高糖），培养三天（密度大概80%）传代。

【注意事项】本细胞标记前与标记后在形态上略有差别，属于正常现象，如下图所示。左图是标记前形态，右图是标记后形态。

   6.      细胞传代所需时间：3天/代 

7.       选择压力：无

8.      体外实验数据：细胞读数为1194 RLU/cell。  

9.      体内实验：肝原位接种细胞3×10⁶，两周后活体荧光成像。