Effect Evaluation Of Conditional Removal Of Germ Cells In Transgenic Mice Model

Reproductive diseases are becoming more and more serious in our society as a result of the pathological changes of reproductive organs or the growth status of reproductive cells.With the maturity of cell culture technology,stem cell transplantation has become one of the therapies with the injection of stem cells.However,there has no ideal animal model for evaluating the effect of germ cell transplantation.In order to solve this problem,our team obtained pSohlh2-Tet3G-TRE 3G-Flag-DTA transgenic mice in Kunming strain by prokaryotic injection.The expression of diphtheria toxin A(DTA)was induced by doxycycline(Dox)in Tet-on expression system.Under the action of promoter Sohlh2,the germ cells are specifically removed.After I got the transgenic mice prepared by Gao Fenglei,I evaluated the removal effect of it.First of all,improve the probability of homozygous transgenic mice by means of inbreeding,then test crossing with wild type Kunming mice,detect the positive rate in offspring;screening homozygous transgenic mice.Secondly,the transgenic animals were obtained by the method of prokaryotic injection,in which the foreign gene was inserted into the genome in a random way.Determine the location of foreign gene by Genome Walking.Then,induct in transgenic mice and effect detection.In the early stage,we tried many kinds of methods,like intraperitoneal injection drug,add drug in drinking water,rectum absorption.By contrast,add drug in drinking water from early pregnant period until the offspring mice grew up,can have the best germ cells removal effect.The drug concentration is 2mg/mL.Induct transgenic mice during pregnancy,at the same time with wild type Kunming pregnant rats as a control experiment.Ten weeks later,the expression of rtTA protein in the tissues was detected by Western blot method.In situ hybridization experiments were performed to detect the transcription sites of DTA and rtTA genes in ovary and testis.Fixed with 4% paraformaldehyde,then contrast morphological changes before and after induced by paraffin section.The expression of rtTA was detected in ovary and testis of transgenic mice after induced by Dox.In situ hybrid ization results show that the target gene was mainly expressed in the basal lamina of seminiferous tubules.Morphologically,the germ cells of transgenic mice induced by Dox were significantly reduced,while there was no significant change in wild-type mice.The whole results show that after the induction of Dox in the transgenic mice,male germ cells were killed and removed.Finally,take C57 neonatal male mice 3-5 days after birth for the preparation of SSCs,transplanted into the seminiferous tubules of male transgenic empty model.Male mice treated busulfan injection to transplant as control.The results show that after the injection of C57 SSCs,the number of luminal cells in the testis increased significantly.In conclusion: steady and hereditary transgenic Kunming mice were obtained.By Genome Walking method we know that the integration of target gene on chromosome 4 of the mouse genome.Add drug in drinking water to transgenic mice after pregnant,the male offspring appeared obvious germ cell clearance.Recover after the transplantation of C57 SSCs.It lays foundation for the following research.