Model Construction Of Hr-knockout Mouse And Its Biological Characteristics Primary Research

Background:The hairless gene(Hr)is a gene containing a zinc finger domain and associated with hair growth.The gene encodes a protein(HR)that is a transcription factor expressed highly in skin and brain,and regulates thyroid hormone receptor.It acts on the hair follicle cells,and its function maintains hair follicle cells proliferation,between the differentiation and apoptosis of the balance,thus it also affects the growth cycle of hair follicles.Lossing hairless gene leads to the hair follicles regeneration hinder,and induces forming a complex skin phenotype.The mutant hairless mice is first discovered in the mice on 1924.The mutation in hairless gene causes hair loss in the skins of mice,but hair follicles formation during postnatal growth are normal in the mutant mice.At present,it is difficult for us to know the mechanism that influences the hair growth.Mouse model affords an excellent approach to investigate the contribution of a particular gene defect to the observed phenotype by targeting the expression of in knock-out mice.Methods:1.The topic that constructs knockout mice by the experimental technology of TALEN.DNA of the offsprings mice are extracted,PCR amplification,sequencing and positive identification.Hr gene knockout mice have been bred stably by our laboratory.2.The Hr knockout mice are used as experimental subjects to observe the morphological and histological changes at different days.3.We select back skin tissues at different ages of the Hr-knockout homozygous mice and wild-type mice as experimental materials.We detect expression of mutant Hr gene in parts of hair follicle structures by the IHC method,testing the expression level of mRNA and HR by RT-PCR and Western blot.Results:1.We have stably bred two kind of knockout mice.The first is that the coding sequence of 86-87 sites delete two bases GT,and the second knockout strain misses AC bases at 88-89 sites.The genetic characteristics of Hr-knockout mice follow the Mendel's law of inheritance.2.The positive mice are identified by DNA sequencing.The offspring of Hr-knockout homozygous mice loss hair postnatal two weeks from abdomen to back,and there is no hair at its life.The hair phenotypes of two knockout mice stains are similar;but Hr-knockout heterozygous mice have normal hair phenotype.we find that the hair follicle structure of knockout mice disrupt at postnatal 21 days,and the skin tissue began to form cysts of small structures at postnatal 28 days.When the knockout mice are 6 months old,their skins fill with irregular cyst sizes.3.Detecting the expression site of HR by IHC,the results show that knockout mice are obviously visible HR expression in the hair growth cycle of 21 day old,28 day old or June age.However,the expression of HR in wild-type mice are visible only during the telogen of hair follicles,there is only little amount of expression in other periods.4.There is no significant difference in the expression of Hr between the knockout mice and the wild mice at postnatal day 7(P7)and P21(P>0.05).It is obviously higher in the knockout mice than it is in the WT at P14(P<0.05),and the difference is significant.However,it is no obvious difference between knockout and wild mice at P28 and P21.But the Hr expression of WT is significant lower than that of knockout mice at P28(P<0.05).The skin tissues of knockout homozygous mice have high levels of HR expression at the hair removal period,but hair follicle structures of wild-type mice only in telogen have expression.The results show that the expression of Hr regulates the hair growth cycle and development mechanism.Conclusions:1.Knockouting Hr can lead to abnormal hair growth.Hr-knockout mice of genetic background is relatively clear.They can be used as animal models for the study of some skin diseases,hair diseases or aging mechanism.2.Hr-knockout mice skin structure have pathological changes significantly at hair shedding stages,and their hair follicles disintegrate.The normal function of Hr gene deletion hinders the hair follicle forming,thus it causes abnormal hair growth.3.The expression of HR regulates the mechanism of hair growth,the high level of HR in skin of Hr mutant mice causes hair loss.The paper will provide experimental material and theoretical basis for studying on the etiology and molecular mechanism of modern hair diseases such as alopecia or alopecia areata.