Phenotypic Analysis Of Klf17 Knockout Mice

Chapter 1:Klf17 expression in mouse tissuesObjective:Sp1/kruppel-like transcription factors(Sp1/KLFs)are a group of zinc finger-containing transcription factors involved in eukaryotic cell transcription.Kruppel-like transcription factor 17(KLF17),a member of Sp1/KLFs,is expressed in human testis,adrenal gland,appendix,endometrium,esophagus,gallbladder,lung and bladder.In mice,the expression of Klf17 was first detected in the testis and ovary,and later in the brain.To date,the expression of Klf17 in other mouse tissues remains unclear.In this chapter,we aim to examine the tissue expression profile of Klf17 in C57BL/6J mice.Methods:1.Tissues from wild-type(WT)C57BL/6J mice were collected and used for extraction of mRNAs.2.Reverse transcription polymerase chain reaction(RT-PCR)was used to analyze the expression of Klf17 mRNA in the tissues.3.Immunohistochemistry(IHC)was used to determine the expression and cellular location of Klf17 protein in the tissues.Results:1.Klf17 mRNA expression was detected in spleen,lung,kidney,stomach,testis,cerebellum,trachea and ovary samples.2.Klf17 protein expression was detected in bronchial goblet cells and vascular smooth muscle cells in the lung,renal tubules in the kidney,chief cells in the stomach and spermatid in the testis.Conclusion:We analyzed KIF17 expression in mouse tissues.We found that Klf17 was expressed in the testis,which is consistent with the previous report.In addition,the expression of Klf17 was also found in the lung,kidney and stomach of mice.These results help to better understand Klf17 expression profile in mice and to guide our studies of Klf17 physiological function in the following chapters.Chapter 2:General phenotypic analysis of Klf17 knockout miceObjective:Unlike most members of the KLF family,the physiological function of KLF17 remains unclear.In human cancers,KLF17 has been reported to inhibit tumor metastasis by blocking tumor epithelial mesenchymal transformation.In mice,Klf17 is the first transcription factor with the zinc finger structure found in germ cells,suggesting a potential function of Klf17 in reproduction.Previous literatures indicate that Klf17 is involved in the formation and differentiation of erythroid cells in developing embryos in zebrafish.In this chapter,we characterized Klf17 knockout(KO)mice and studied the general phenotype of the mice to understand the physiological function of Klf17 in embryonic development and postnatal survival.Methods:1.DNAs were isolated from biopsy samples and used in PCR to genotype Klf17 KO mice.2.RT-PCR was used to analyze Klf17 mRNA in tissues from WT mice and Klf17 KO mice.3.IHC was used to determine Klf17 protein expression in Klf17 KO mice.4.Heterozygous male and female Klf17 KO mice were mated and genotyping was done in the offspring to determine genotype distribution.5.Necropsy was performed and histological analysis was done in hematoxylin and eosin(H&E)-stained tissue sections from WT and Klf17 KO mice.6.Postnatal survival was monitored up to 600 days in WT and Klf17 KO mice and survival curves were analyzed.7.Postnatal growth was measured in male and female Klf17 KO mice and compared with that in WT mice.8.Blood cell counts were analyzed in WT and Klf17 KO mice by a blood cell analyzer.Results:1.Klf17 mRNA and Klf17 protein were not detected in Klf17 KO mice.2.Genotypes among offspring from heterozygous of Klf17 KO male and female mice showed the Mendelian distribution pattern.3.No apparent abnormalities were observed in major organs,including heart,liver,spleen,lung,stomach,small intestine and testis,from Klf17 KO mice,compared to those from WT mice.4.The long-term survival of Klf17 KO mice was not different from that of WT mice.5.Postnatal body weight gains of Klf17 KO mice were not different from those of WT mice.6.Compared with male WT mice,male Klf17 KO mice had higher red blood cell counts,higher levels of hemoglobin and hematocrit,and lower mean corpuscular volumes.Male Klf17 KO mice also had higher white blood cell counts and lower platelet,neutrophil and eosinophil counts.In contrast,those differences were not observed between female Klf17 KO and WT mice.Conclusion:Our results showed that Klf17 deficiency has no significant effects on mouse embryo development and postnatal survival,whereas the blood cells of the mice were affected,resulting in higher red blood cell counts,higher levels of hemoglobin and hematocrit.In addition,the effect of Klf17 deficiency on blood cells of mice was shown in male mice,but not in female mice,suggesting that the effect might be related to sex hormones.These results suggest a potential role of Klf17 in red blood cell production.Chapter 3:Effects of Klf17 deficiency on the pregnant mouse uterusObjective:Pre-eclampsia is a major disease in pregnancy,affecting 5-8%of pregnant women in the world.Severe preeclampsia causes maternal and fetal deaths.Impaired spiral artery remodeling is a crucial pathological mechanism of preeclampsia.Corin,a serine protease,promotes spiral artery remodeling in the pregnant uterus.Previous studies in our laboratory showed that KLF17 directly bound to the CORIN promoter to regulate corin expression in cultured human uterine cells.In this chapter,we used Klf17 KO mice to study the regulation of corin expression by Klf17 in the pregnant uterus,and to analyze the phenotype in pregnant Klf17 KO mice.Methods:1.RT-PCR and real-time PCR were used to examine Corin mRNA expression in the pregnant uterus from WT and Klf17 KO mice at different gestational stages.2.The fertility of male and female Klf17 KO mice was analyzed by different mating strategies.3.H&E stained and IHC were used to analyze uterine spiral artery remodeling in pregnant WT and Klf17 KO mice.4.Blood pressure was determined by a tail-cuff method in WT and Klf17 KO mice at different gestational stages.5.Urinary protein levels in WT and Klf17 KO mice at different gestational stages were measured by a Bradford method.6.H&E,PAS and Masson staining were used to determine the morphological changes in glomeruli from WT and Klf17 KO mice at different gestational stages.7.Glomerular red blood cell counts were analyzed in kidney sections from WT and Klf17 KO mice at different gestational stages.Results:1.Corin mRNA expression was detected in the heart,but not the pregnant uterus,in Klf17 KO mice.2.The litter size from Klf17 KO mice was smaller than that from WT mice(4.1±0.3 vs.7.1±0.4;p<0.001).Moreover,the litter size of Klf17 KO females mated with WT males was also smaller(3.3±0.3 vs.7.3±0.5;p<0.001).By comparison,no significant difference was observed in the litter size between WT females mated with Klf17 KO or WT males(6.5±0.2 vs.7.3±0.5;p=0.1294).3.Approximately 32%embryos from Klf17 KO female mice died during the gestational period.4.In Klf17 KO mice,shallow trophoblast invasion and impaired spiral artery remodeling were observed in the pregnant uterus.5.Increased blood pressure occurred at late gestational stages in Klf17 KO mice.6.Increased urinary protein levels were detected at late gestational stages in Klf17 KO mice.7.Glomerular erythrocyte numbers were lower in pregnant Klf17 KO mice than those of control WT mice.ConclusionUterine corin expression in WT mice gradually increased during pregnancy.In the pregnant uterus of Klf17 KO mice,corin expression was not detected,suggesting that Klf17 is a key transcription factor for corin up-regulation in the pregnant uterus.Deficiency of Klf17 prevents Corin up-regulation in pregnant uterus and leads to a pre-eclampsia-like phenotype in mice:increased blood pressure in late pregnancy,increased urinary protein levels,fetal growth restriction and demise,and impaired spiral artery remodeling.