一種高效省時的利用磷酸鈣轉染DNA的改進方法

黃進++傅吉強++張慶華

摘 要 對傳統磷酸鈣轉染方法進行改進，即在經胰酶消化處理成懸浮狀態的293T細胞懸液中直接加入磷酸鈣轉染試劑，而不需待其貼壁后再加轉染試劑進行轉染.通過比較懸浮和貼壁狀態下293T細胞的轉染效率、細胞增殖和熒光強度等指標，發現這兩種轉染方法的各項指標沒有顯著性差異.結果表明，293T細胞在懸浮狀態下進行磷酸鈣轉染與傳統的貼壁轉染能夠達到同等效果，在用胰酶消化的單細胞懸液中直接加入磷酸鈣轉染試劑進行轉染，不影響細胞的轉染效果，節省了操作步驟，縮短了操作時間.

關鍵詞 293T細胞；磷酸鈣；貼壁狀態；懸浮狀態；細胞轉染

中圖分類號 Q813.1 文獻標識碼 A 文章編號 10002537（2016）01001806

An Efficient and TimeSaving Method for DNA Transfection Using Calcium Phosphate

HUANG Jina， Fu Jiqiangb， ZHANG Qinghuaa*

（a.School of Life Science and Technology， b.School of Medicine， Tongji University， Shanghai 200092， China）

Abstract The use of calcium phosphate for the transfection of adherent cells was a traditional and popular yet complicated and timeconsuming method. In this paper， this traditional method is further improved through the direct addition of calcium phosphate transfection reagent into the trypsinized 293T cell suspension solution， instead of adding transfection reagent after the cells were adhered to the bottom. By comparison with such indicators as transfection efficiency， cell proliferation and fluorescence intensity of 293T cells in suspended status and adherent status， we found no significant difference between the two transfection methods with regard to these indicators. Based on the results from this work， the use of calcium phosphate in transfecting 293T cells in suspended status could be same efficient and effective as the traditional adherent transfection method. With the transfection efficiency unaffected， our method of directly adding calcium phosphate transfection reagent into the trypsinized singlecell suspension solution is not only experimentally simplified in procedure but also timesaving in operation.

Key words 293T cells； calcium phosphate； adherent status； suspended status； cell transfection

目前有多種細胞轉染方法，例如脂質體轉染法[1]、基因鳥槍法、電穿孔法[2]、顯微注射法[3]和磷酸鈣共沉淀法等.脂質體轉染法雖然操作簡單，轉染效率較高，但脂質體價格昂貴，且細胞毒性較大；基因鳥槍法速度快，簡單易行，成本較低，但不適用于真核細胞；電穿孔法轉染效率高，但所需儀器較貴，對細胞損傷較大；顯微注射法整合效率高，不需載體，但需精密儀器，技術難度大，且價格昂貴；磷酸鈣轉染所需材料價格低廉，操作簡單，轉染效率高，適用范圍廣，且細胞毒性較低.所以，在很多DNA轉染細胞實驗中，多用磷酸鈣作為轉染試劑.

利用磷酸鈣將攜帶有外源基因的質粒DNA轉染到哺乳動物細胞，是一種操作簡單且應用廣泛的方法[4].磷酸鈣共……