表皮细胞的转染实验技巧
表皮细胞广泛遍布于身体，正常的表皮细胞较难转染，尤其是使用基于脂质体技术的转染试剂。我们使用电转(Amaxa)方法转染正常人的结肠表皮细胞并得到了65%的GFP标记细胞。非常感谢SignaGen,现在我们使用GenJet Ver II可以成功转染正常人的结肠表皮细胞并且转染效率显著提高至75%。
如下是使用GenJet Ver II(Cat#SL100489)转染表皮细胞的简单步骤：
1、转染时确保表皮细胞达到85%的融合度，并且保证细胞新鲜、健康。
2、对于6孔板，用不含血清的DMEM分别稀释1.0µg,DNA及3.0µl,GenJet Ver.II（Cat＝SL100489）.将稀释好的转染试剂加入DNA中，室温下放置15分钟以形成转染复合物。
3、将转染复合物直接加入表层细胞中：6孔板，每孔含1.0ml培养基，在血清/抗生素存在下，转染进行。
4、在转染进行5小时后，清除转染复合物并换成正常生长培养基。
5、转染后的24-48小时，检测转染基因的表达情况。
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.
