Construction And Functional Study Of SIK2 Knock-out Mice Model

To further study the function of SIK2 protein in vivo,we are to construct SIK2 knockout mouse model.It mainly includes the function of SIK2 in ionizing radiation injury,immune and immune damage repair and T cell antigen receptor(TCR)gene rearrangement,and lay a solid foundation for the further study of SIK2 function.First,construct the ES cell targeting vector with two Loxp loci,and then transfected into ES,two Loxp loci were integrated into the genome by homologous recombination,respectively located in the SIK2 gene exon 2 and 3 on both sides of the intron,screening positive clones of ES with two Loxp loci,blastocyst cavity positive ES cloning of microinjection into Albino B6 mi ce,and transplanted into pseudopregnant uterus in female mice,the chimeric mice were 3 weeks old when the cage to identify the genotype is SIK2 Lox Pl-2+/-,the genotype of mice after purification with E??-Cre+/+ tool mouse hybrid,hybrid screening of SIK2Loxp1-2+/-Cre+/-mice,the mice with WT type selection of SIK2Loxp3+/-Cre-/-(SIK2+/-)mice,and SIK2+/-genotype in male mice hybridization,SIK2-/-genotype screening of mice.Subsequently,SIK2+/+(WT)and SIK2+/-(KO)mice were used to study the function.WT and KO mice were treated with 8 Gy 60 Co gamma ray irradiation,respectively.The growth and survival of the patients were observed and recorded every day,and their survival rate was calculated in the 30 days.The mice were crossed with SIK2+/-genotype,and 13.5 days after pregnancy,the embryos were isolated and WT and KO MEFs cells were isolated.The difference of radiosensitivity between the two groups was observed by clone formation experiment.WT and KO mice red blood cells,white blood cells,platelets and hemoglobin content through the blood routine analysis,and bone marrow nucleated cell count,to observe the effects of SIK2 knockdown on hematopoietic function.WT and KO mice were irradiated with 6 Gy 60 Co gamma ray irradiation at one time,and then sacrificed at 1,3 and 7d after irradiation,and then the femur was fixed.The number of bone marrow nucleated cells was observed by HE staining.The use of immune cells,flow cytometry was used to detect WT and KO in peripheral blood,thymus,bone marrow and spleen tissue in the proportion of number of T,B cell subgroup analysis,combined with the observation of pathological changes in the immunohistochemical HE staining of mouse SIK2 gene knockout on the i mmunity effect.Once again,the WT and KO mice were irradiated with 6 Gy 60 Co gamma rays,and then were sacrificed at the time of 1,3 and 7d,and then the thymus,bone marrow and spleen tissue were taken.After fixation,the damage of immune organ was observed by HE staining.SIK2+/+ and SIK2+/-in mice thymus extract,thymocyte DNA,designed for the TCR? site V?8-D?2J?2 recombinant fragment specific probes by Southern blot detection of WT and KO mice TCR beta site V(D)J whether there are differences betwe en the recombinant.The results showed that ES cells transfected with ES targeting vector after two Loxp loci were successfully integrated into the genome,screened positive clones of ES.The blastocyst microinjection in the uterine cavity,transplantation of pseudopregnant female mice received SIK2 Lox Pl-2+/-chimeric mice.The chimeric mice were purified with E??-Cre+/+ mouse hybrid screening tool,SIK2Loxp1-2+/-Cre+/-mice,mice and the hybrid type WT mice received SIK2Loxp3+/-Cre-/-(SIK2+/-)mice,the SIK2+/-genotype in male mice without hybridization,screened SIK2-/-genotype of mice,but the 15 d is normal in embryonic development there may exist SIK2 knockout embryo after death.The survival rate of mice and the experimental results of colony formation showed that the radiosensitivity of SIK2 knockout mice and MEFs cells was enhanced.The blood routine analysis showed KO mice red blood cells,white blood cells,platelets and hemoglobin content were significantly lower than that of WT.The results of bone marrow nucleated cells showed that the number of nucleated cells in bone marrow of KO mice was less than WT.KO mice at 1D after irradiation,3D mice bone marrow nucleated cell number was significantly reduced to seventh days,the number of bone marrow cells has increased significantly,showed that knockdown of SIK2 can inhibit the hematopoietic system damage repair induced by radiation in a certain extent.Flow cytometry results showed that KO CD4+CD8-and CD4-CD8+ T cells in the thymus were significantly reduced,CD4+CD8+ T cells were significantly increased;reduce the content of CD3+ T cells in peripheral blood,CD4+CD8-,CD4-CD8+ content of T cells decreased,whereas immature CD4-CD8-T cells were significantly increased;the bone marrow Ig M+B220+ B cells and B220+CD43-content decreased however,immature progenitor cells B B220+CD43+ B cells showed no obvious change,there may be a knockout of SIK2 resulted in the differentiation of B cells in other subtypes of mature stage;content of CD4+CD8-,CD4-CD8+ single positive T cells and the content of Ig M+B220+ B cells in the spleen was significantly reduced.HE staining of tissue sections showed that the pathological structure of thymus,spleen and bone marrow of SIK2+/-(KO)mice was not significantly different from that of SIK2+/+(WT).However,after irradiation,the thymus,spleen and bone marrow of SIK2+/-(KO)mice showed that the damage was more serious than that of WT,and the recovery was slower.Finally,we also found that the TCR beta gene fragment of V8-D2J2 was not detected in DNA thymocytes of SIK2+/-mice.Experiments show SIK2 gene knockout mice was successfully constructed by Cre-Lox P recombinase system,but not yet been completely SIK2-/-knockout mice,which may exist in the embryonic lethality.SIK2 knockout mice and MEFs cell radiosensitivity enhancement after hematopoietic function;mice;lymphocyte maturation blocked;immune system damage caused by radiation weakened;failure of mouse TCR beta site V-DJ gene rearrangement.