LOXP-DsRed-LOXP系统的构建及其应用

Construction and application of LOXP-DsRed-LOXP system

目的构建荷载LOXP-Ds Red-LOXP系统的细胞,并探索该系统在体内外检测Cre酶活性中的应用。方法构建稳转株MCA38^(LOXP-Ds Red-LOXP)并体外验证检测Cre酶效能。将Balb/c裸鼠随机分为两组(n=10),分别在结直肠黏膜下移植MCA38^(Cre-Luciferase)与MCA38^(Luciferase)细胞株,两周后取灌肠液作用于MCA38^(LOXP-Ds Red-LOXP),验证该系统体内检测Cre酶效能。APC^(LOXP/LOXP)小鼠随机分为两组(n=20),分别使用可表达Cre酶及荧光素酶(Luciferase)的慢病毒颗粒(CL组)与仅表达Luciferase的慢病毒颗粒(L组)行小鼠结直肠黏膜下注射,感染后第3天及第3周末取灌肠液检测Cre酶活性及表达情况,同时行小鼠活体成像检测Luciferase表达情况,验证该系统在判断Cre酶活性与预测基因修饰成功率中的应用。结果 Cre重组酶作用于MCA38^(LOXP-Ds Red-LOXP48) h后,红色荧光明显减弱。裸鼠灌肠液作用于MCA38^(LOXP-Ds Red-LOXP)细胞株48 h后,MCA38^(Cre-Luciferase)组红色荧光明显减弱。CL组模型鼠病毒注射后第3天检出16只小鼠荧光素酶及灌肠液Cre酶阳性,第3周末8只小鼠荧光素酶及灌肠液Cre酶阳性。L组模型鼠病毒注射后第3天检出17只小鼠荧光素酶阳性而灌肠液Cre酶均阴性,第3周末检出8只小鼠荧光素酶阳性,灌肠液Cre酶均阴性。结论成功构建Cre酶检出系统MCA38^(LOXP-Ds Red-LOXP),实现了体内、外无创性检测Cre酶活性及表达情况,并以此评价APC^(LOXP/LOXP)小鼠结直肠黏膜基底干细胞病毒感染效率及基因修饰情况。

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 MCA38^LOXP-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）. MCA38^Cre-Luciferase and MCA38^Luciferase 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 MCA38^LOXP-DsRed-LOXP cells treated with nude mice enema for more than 48 hours was significantly faded in MCA38^Cre-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 APC^LOXP/LOXP mice.