LOXP—DsRed—LOXP系統的構建及其應用

宋丹++鄭勇斌+童仕倫++肖曠+楊超++孫偉+秦凱迪

[摘要] 目的 構建荷載LOXP-DsRed-LOXP系統的細胞，并探索該系統在體內外檢測Cre酶活性中的應用。 方法 構建穩轉株MCA38LOXP-DsRed-LOXP并體外驗證檢測Cre酶效能。將Balb/c裸鼠隨機分為兩組（n=10），分別在結直腸黏膜下移植MCA38Cre-Luciferase與MCA38Luciferase細胞株，兩周后取灌腸液作用于MCA38LOXP-DsRed-LOXP，驗證該系統體內檢測Cre酶效能。APCLOXP/LOXP小鼠隨機分為兩組（n=20），分別使用可表達Cre酶及熒光素酶（Luciferase）的慢病毒顆粒（CL組）與僅表達Luciferase的慢病毒顆粒（L組）行小鼠結直腸黏膜下注射，感染后第3天及第3周末取灌腸液檢測Cre酶活性及表達情況，同時行小鼠活體成像檢測Luciferase表達情況，驗證該系統在判斷Cre酶活性與預測基因修飾成功率中的應用。 結果 Cre重組酶作用于MCA38LOXP-DsRed-LOXP48 h后，紅色熒光明顯減弱。裸鼠灌腸液作用于MCA38LOXP-DsRed-LOXP細胞株48 h后，MCA38Cre-Luciferase組紅色熒光明顯減弱。CL組模型鼠病毒注射后第3天檢出16只小鼠熒光素酶及灌腸液Cre酶陽性，第3周末8只小鼠熒光素酶及灌腸液Cre酶陽性。L組模型鼠病毒注射后第3天檢出17只小鼠熒光素酶陽性而灌腸液Cre酶均陰性，第3周末檢出8只小鼠熒光素酶陽性，灌腸液Cre酶均陰性。 結論 成功構建Cre酶檢出系統MCA38LOXP-DsRed-LOXP，實現了體內、外無創性檢測Cre酶活性及表達情況，并以此評價APCLOXP/LOXP小鼠結直腸黏膜基底干細胞病毒感染效率及基因修飾情況。

[關鍵詞] Cre酶；DsRed熒光蛋白；LOXP-DsRed-LOXP系統；結直腸癌

[中圖分類號] R735 [文獻標識碼] A [文章編號] 1673-7210（2017）11（c）-0004-05

Construction and application of LOXP-DsRed-LOXP system

SONG Dan ZHENG Yongbin TONG Shilun XIAO Kuang YANG Chao SUN Wei QIN Kaidi

Department of Gastrointestinal Surgery， Renmin Hospital of Wuhan University， Key Laboratory of Hubei Province for Digestive System Disease， Hubei Province， Wuhan 430060， China

[Abstract] Objective To Establish a cell line contains the LOXP-DsRed-LOXP system， in order to study its application in detecting the activity of Cre recombinase in vitro and in vivo. Methods MCA38LOXP-DsRed-LOXP cells were established to validate its ability of detecting the activity of Cre recombinase in vitro. Balb/c nude mice were randomly divided into two groups （n=10）. MCA38Cre-Luciferase and MCA38Luciferase cells were transplanted in colorectal mucosa respectively and then collected enema two week later to validate its ability of detecting the activity of Cre recombinase in vivo. APCLOXP/LOXP mice were randomly divided into two groups （n=20）. Lentivirus expressing Cre and luciferase enzyme or expressing luciferase only were injected to colorectal mucosa respectively. Enema were collected at the 3rd day and 3 weeks later the activity of Cre recombinase was detected. Meanwhile， the in vivo imaging of mice was applied to detect the expression of luciferase. The application of this system in detecting the activity of Cre recombinase and predicting the success rate of gene modification was verified. Results The results showed that the red fluorescence was significantly faded in MCA38 LOXP-DsRed-LOXP cells which was modificated by Cre recombinase for more than 48 hours in vitro. Besides， the red fluorescence of MCA38LOXP-DsRed-LOXP cells treated with nude mice enema for more than 48 hours was significantly faded in MCA38Cre-Luciferase group. In group CL， Luciferase and Cre recombinase were positive in 16 mice at the 3rd day and 8 mice at the third weekend after injecting Lentivirus. In group L， Luciferase was positive in 17 mice at the 3rd day and 8 mice at the third weekend after injecting Lentivirus， while Cre recombinase was negative in all. Conclusion LOXP-DsRed-LOXP system are successfully constructed. The in vivo and in vitro non-invasive detection of Cre recombinase activity and expression was achieved. By which it could evaluate the efficiency of virus infection and gene modification of colorectal mucosal basal stem cells in APCLOXP/LOXP mice.endprint

[Key words] Cre recombinase； DsRed fluorescent protein； LOXP-DsRed-LOXP system； Colorectal cancer

Cre/Loxp系統所介導的重組反應僅依賴于Cre酶及其識別位點Loxp序列，不需要消耗能量及其他輔助因子的參與，同時沒有種屬特異性，因而被廣泛應用于基因修飾及其功能鑒定的研究中[1-3]。……