Preliminary Study Of Conditional Cell Depletion In Uterus

Conditional cell depletion can specifically ablate the cell that we expect to remove.It can help us to study the functions of cells and the interactions between various cells in vivo.To achieve this goal,some methods was gradually developed,such as physical ablation,cytotoxic antibodies,chemicals,thymidine kinase,nitroreductase.However,these methods have many shortcomings.In recent years,a new cell depletion method has been developed.It is base on transgenic expression of a diphtheria toxin receptor?DTR?in mouse cells and application of diphtheria toxin?DT?.The uterus mainly consists of the endometrium,stroma,epithelium,and various immune cells.In this experiment,PR^Cre/+,Amhr2^Cre/+,and Ltf^Cre/+mice were mated with pCAG-STOP-DTR-2A-EGFPmicetoobtainPR^Cre/+/pCAG-STOP-DTR-2A-EGFP,Amhr2^Cre/+/pCAG-STOP-DTR-2A-EGFP,Ltf^Cre/+/pCAG-STOP-DTR-2A-EGFP mice.They were designated as PR-DTR,Amhr2-DTR and Ltf-DTR,respectively.Using immunohistochemistry,and Western blotting detected the specific expression of DTR,results showed that DTR specific expressed in the different parts of the three obtained mouse uteri.The results of intraperitoneal injection of DT in pCAG-STOP-DTR-2A-EGFP mice showed that injected with 10ng DT for two consecutive days,mice died after 1-3 days;injected with 5ng DT for three consecutive days,mice deteriorated or died;mice were injected with 5ng DT for two consecutive days,it showed that the maximum tolerated dose of DT was 10 ng;mice injected with 1ng DT for three consecutive days showed no effect.PR-DTR,Ltf-DTR,DTR and WT females mated with healthy adult males and injected with 1ng DT daily at the second and third days of pregnancy.The uterus was dissected on the fourth day of pregnancy,and observed its morphology.In the PR-DTR mice,the uterus was swollen and redness,but there was no significant change in the uterus of the Ltf-DTR mice compared with the wild-type mice.The abnormal cavity in the uterus of PR-DTR mice was observed by HE staining,but Ltf-DTR mice had no change.Then the expression of FOXA2 in different mouse uterus was detected by immunohistochemistry.The above results demonstrate that DT plays a role in the PR-DTR mice uterus by depleting cells expressing DTR.The Ltf-DTR females injected DT and drew materials after 12 hours.We observed that the uterus is swollen.HE staining was used to observe the internal morphology.The internal epithelial cells were loosely arranged and the glandular epithelium fell off.It was demonstrated that after DT administration 12 h DT could act in the uterus of Ltf-DTR female mice.By above experimental results,we concluded that endometrium,stromal and epithelial cells can be knocked out of PR-DTR mice,and Ltf-DTR mice can knock out epithelial cells.This shows that these transgenic mice can be used to study the occurrence and development of cells in different regions of the uterus,the physiological functions,the interactions between cells in the uterus,depletion of Epithelial Cells by Ltf-DTR Mice can be used to study the physiological functions in the uterus during the menstrual cycle,and can also be used as disease models to study various uterine diseases.