Construction And Analysis Of The Aicardi-Goutieres Syndrome Associated Gene SAMHD1 Knock-out Rat Model

Background & Objective: Aicardi-Goutières syndrome(AGS)is a rare hereditary nervous system disease that often occurs in newborns and it is considered an autoimmune disease.The clinical manifestations of AGS are subacute encephalomyelitis with brain atrophy,intracranial calcification,white matter lesions,cerebrospinal fluid lymphocytosis,and abnormal elevation of interferon alpha(IFN-?)levels.It was found that mutations in the SAMHD1(Sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1)gene can cause AGS.In 2013,two independent research groups established a SAMHD1-deficient mouse model and found that type I interferon-inducible gene expression was up-regulated in peritoneal or bone marrow-derived macrophages of SAMHD1-deficient mice,but it did not show serious autoimmune disease symptoms.In 2015,Kasher PR established a SAMHD1-deficient zebrafish model that can present brain symptoms similar to human AGS.However,zebrafish is not mammals and it is not well suited for studying the molecular mechanisms of pathogenesis and targeted therapies of human AGS.The rat nervous system is similar to humans and the SAMHD1 gene-deficient rat is likely to be an ideal animal model for studying human AGS.The aim of this study is to establish the SAMHD1-knockout rat model and observe whether this model has symptoms similar to human AGS.This study can lay the foundation for further study of AGS pathogenesis and targeted therapy.Methods1.Construction of SAMHD1-knockout rat by CRISPR/Cas9 technology and its PCR identificationa)sgRNA design and its construction: The sgRNA is constructed which acts on the fourth exon of the SAMHD1 gene.b)Pronuclear injection and transplantation: superovulation treatment of rats;injection of sgRNA into fertilized eggs,transplantation for obtaining F0 generation rats.c)Identification of F0 generation rat and breeding: knockout(KO)positive rats were verified by PCR and DNA sequencing;positive F0 generation rats and SD rats were backcrossed.d)Identification of F1 generation rat: Positive F1 rats were verified by PCR and DNA sequencing.e)F1 hybrid rats were bred in cages,the postnatal rats were identified by PCR and DNA sequencing for obtaining the F2 generation which were SAMHD1-knockout homozygous rats.Homozygous F2 generation rat were bred in cages to generate a number of SAMHD1-knockout homozygous rats for follow-up studies.2 Detection of SAMHD1 expression in various organs and tissues of SAMHD1-deficient ratsDetection of SAMHD1 in various organs and tissues by western blot3 Is there a lesion in the brain of SAMHD1-deficient rats?a)Magnetic resonance imaging(MRI)was used to detect brain atrophy,intracranial calcification and white matter lesions in SAMHD1-deficient rats.b)The Morris water maze method was used to detect the navigational and spatial exploration capabilities of SAMHD1-deficient rats.4 Are SAMHD1-deficient rats with symptoms of autoimmune disease with abnormally elevated type I interferon?Peripheral blood of SAMHD1-deficient rats was extracted for about 8 weeks and IFN-? and ? were detected by enzyme linked immunosorbent assay(ELISA).5 What are the levels of type I interferon expression in organs and tissues of SAMHD1-deficient rats?The organs and tissues of SAMHD1-deficient rats were taken for about 8 weeks.Immunohistochemical staining was used to detect whether IFN-? and ? were highly expressed in all organs and tissues.Result1.PCR and sequencing results showed that 3 KO positive F0 rats were obtained.The 24 th is the-184 bp knockout and the 25 th is the-346 bp knockout.2.PCR and sequencing results showed that F1 generation 32 th and 33 th were-184 bp knockout heterozygous rats.53 th,56 th and 57 th were-346 bp knockout heterozygous rat.3.F1 generation knockout heterozygotes were used for cage breeding and a total of 9 F2 generation rats were obtained.3 Mesozygous(SAMHD1-/-)(85th,86 th,88th),4 heterozygous(SAMHD1+/-)(87th,89 th,90th,91th),2 wild type(92th,93th).4.Western Blot results showed that the SAMHD1 gene was knocked out and could not be expressed in all organs and tissues of the SAMHD1-deficient homozygous rat.5.Magnetic resonance imaging showed no significant brain atrophy,intracranial calcification and white matter lesions in 8 weeks of SAMHD1-deficient rats.6.ELISA results showed that compared with wild-type rats,IFN-? and ? were significantly increased in peripheral blood serum of SAMHD1-deficient rats for 8 weeks.7.Immunohistochemistry and qRT-PCR results showed that IFN-? and ? were highly expressed in organs and tissues of SAMHD1-deficient rats for 8 weeks.ConclusionThe SAMHD1 knockout rat model was successfully constructed and found to have high expression of type I interferon associated with Aicardi-Goutieres syndrome.