表皮細胞的轉染實驗技巧

表皮細胞廣泛遍布于身體，正常的表皮細胞較難轉染，尤其是使用基于脂質體技術的轉染試劑。我們使用電轉(Amaxa)方法轉染正常人的結腸表皮細胞并得到了65%的GFP標記細胞。非常感謝SignaGen,現在我們使用GenJet Ver II可以成功轉染正常人的結腸表皮細胞并且轉染效率顯著提高至75%。

如下是使用GenJet Ver II(Cat#SL100489)轉染表皮細胞的簡單步驟：

1、轉染時確保表皮細胞達到85%的融合度，并且保證細胞新鮮、健康。

2、對于6孔板，用不含血清的DMEM分別稀釋1.0μg,DNA及3.0μl,GenJet Ver.II（Cat＝SL100489）.將稀釋好的轉染試劑加入DNA中，室溫下放置15分鐘以形成轉染復合物。

3、將轉染復合物直接加入表層細胞中：6孔板，每孔含1.0ml培養基，在血清/抗生素存在下，轉染進行。

4、在轉染進行5小時后，清除轉染復合物并換成正常生長培養基。

5、轉染后的24-48小時，檢測轉染基因的表達情況。

Transfection of epithelial cells.

Epithelial cells are found throughout the body, from skin to glandular formations within tissues. In vivo these cells are attached to a three dimensional basement membrane matrix. Normal epithelial cell is extremely hard to transfect especially to liposome based transfection reagents. We ever used electroporation (Amaxa) to transfect normal human colonic epithelial cells and got 65% GFP+ cells. Thanks to SignaGen, now we have successfully transfected normal human colonic epithelial cell with GenJet Ver. II with up to 75% GFP+ efficiency. The following is a brief protocol for transfecting epithelial cell with GenJet Ver. II (Cat # SL100489):

1. Grow epithelial cell ~85% confluency at time of transfection. Epithelial cells must freshly prepared and healthy. 

2. For 6-well plate, dilute 1.0 μg of DNA and 3.0 μl of GenJet Ver. II (Cat # SL100489) per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes.

3. Add transfection complex to epithelial cells directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.

4. Remove transfection complex 5 hours after transfection and change back to normal growth medium.

5. Check transgene expression 24~48 hours post transfection.