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.
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