細(xì)胞轉(zhuǎn)染小技巧
表皮細(xì)胞的轉(zhuǎn)染：
表皮細(xì)胞廣泛遍布于身體，正常的表皮細(xì)胞較難轉(zhuǎn)染，尤其是使用基于脂質(zhì)體技術(shù)的轉(zhuǎn)染試劑。我們使用電轉(zhuǎn)(Amaxa)方法轉(zhuǎn)染正常人的結(jié)腸表皮細(xì)胞并得到了65%的GFP標(biāo)記細(xì)胞?，F(xiàn)在我們使用GenJet Ver II可以成功轉(zhuǎn)染正常人的結(jié)腸表皮細(xì)胞并且轉(zhuǎn)染效率顯著提高至75%。
如下是使用GenJetVer II(Cat#SL100489)轉(zhuǎn)染表皮細(xì)胞的簡單步驟：
1、轉(zhuǎn)染時確保表皮細(xì)胞達(dá)到85%的融合度，并且保證細(xì)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(zhuǎn)染試劑加入DNA中，室溫下放置15分鐘以形成轉(zhuǎn)染復(fù)合物。
3、將轉(zhuǎn)染復(fù)合物直接加入表層細(xì)胞中：6孔板，細(xì)胞轉(zhuǎn)染小技巧 每孔含1.0ml培養(yǎng)基，在血清/抗生素存在下，轉(zhuǎn)染進(jìn)行。
4、在轉(zhuǎn)染進(jìn)行5小時后，清除轉(zhuǎn)染復(fù)合物并換成正常生長培養(yǎng)基。
5、轉(zhuǎn)染后的24-48小時，檢測轉(zhuǎn)染基因的表達(dá)情況。
Transfection of epithelial cells.
Epithelial cells are found throughout the body, from skin toglandular formations within tissues. In vivo these cells are attached to athree dimensional basement membrane matrix. Normal epithelial cell is extremelyhard to transfect especially to liposome based transfection reagents. We everused electroporation (Amaxa) to transfect normal human colonic epithelial cellsand got 65% GFP+ cells. Thanks to SignaGen, now we have successfullytransfected normal human colonic epithelial cell with GenJet Ver. II withup to 75% GFP+ efficiency. The following is a brief protocol for transfectingepithelial cell with GenJet Ver. II (Cat # SL100489):
1. Grow epithelial cell ~85% confluency at timeof 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 todiluted DNA and let transfection complex formed at RT for 15 minutes.
3. Add transfection complex to epithelial cells directly. Transfection isconducted in presence of serum/antibiotics with transfection volume of 1.0 mlper well of 6-well plate.
4. Remove transfection complex 5 hours after transfection and change back tonormal growth medium.
5. Check transgene expression 24~48 hours post transfection.
