Study Of Homo HtrA1^L364P Transgenic Mice Construction,Phenotype Analysis And The Preliminary Pathogenic Mechanism

Objective:To construct human HtrA1^L364P transgenic mice(Mut-hHtrA1^L364Pmice)and study their phenotype and pathogenesis.Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy(CARASIL)is a rare autosomal recessive cerebrovascular disease(CSVD).The clinical manifestations were stroke,cognitive decline,pseudobulbar palsy,early baldness and recurrent low back pain.Brain magnetic resonance imaging showed multiple cerebral infarction and extensive subcortical demyelination of white matter.Arteriosclerosis of small arteries,vitreous lesions of the medial membrane,fibrosis of the intima and elastic layer,and concentric stenosis of the lumen were seen in the brain tissue of autopsy patients.The global incidence of the disease is low.There are more than 50 cases reported in the literature.The pathogenic gene of the disease was identified as high temperature requirement A1(HtrA1)by Hara,a Japanese scholar.In 2007,our group reported for the first time a CARASIL family in China.Its clinical features were consistent with the symptoms of CARASIL.Sequencing results showed that T1091C mutation occurred in the HtrA1gene,resulting in the mutation of leucine(Leu,L)to proline(Pro,P)at 364 loci.At present,there is no successful CARASIL model animal at home and abroad,which also limits the study of CARASIL.The aim of this study is to construct model animals based on HtrA1 gene mutation sites found in the reported families,and then to study the pathological typing and pathogenesis of the disease.Transgenic mice are one of the most commonly used disease models.In this study,we intend to use CRISPR/Cas9 technology to insert human HtrA1^L364P signal-free peptide sequence after HtrA1 gene signal peptide in mice and make it fused and expressed.On the one hand,the insertion of human HtrA1^L364P can knock out the background HtrA1 of mice.Through phenotypic analysis,we can study whether human HtrA1^L364P transgenic mice cause CARASIL disease.On the other hand,when human HtrA1^L364P was fused with mice HtrA1 signal peptide,not only the expression and localization of HtrA1 signal peptide would not change,but also the regulatory mechanism of its 5'end would not be affected.Therefore,it can better simulate the real situation of human disease.By establishing human HtrA1^L364P knock-in transgenic mice(named Mut-hHtrA1^L364Pmice),the pathogenic mechanism of HtrA1^L364P can be analyzed,which can be used as research models of related diseases such as CARASIL and CSVD.Methods:The study was divided into two parts.Part ?:Construction and identification of Mut-hHtrA1^L364P miceFirst,the Donor vector is constructed.The CDS,protein and genome sequences of human and mice HtrA1 were retrieved to identify their signal peptide regions.According to the location of HtrA1 protein signal peptide in mice,the target region of small guide RNA(sg RNA)was selected.In this region,three sg RNAs were designed and co-transfected with Cas9 to verify the intracellular activity of sg RNA by T7E1digestion,so as to select the optimal sg RNA and clone the left and right homologous arms accordingly.At the same time,human HtrA1 template was obtained and site-directed mutagenesis was carried out.The mutant sequence selected CCG codon that expressed proline efficiently in mice.The region of human HtrA1 containing no signal peptide was amplified by PCR method,and the human HtrA1 was mutated to obtain the human HtrA1^L364P sequence,which was inserted into the left and right homologous arms of the clone to obtain the Donor vector knocked in by CRISPR/Cas9.Secondly,after superovulation,the healthy female mice were closed in cages,and the oviducts of the mice with vaginal suppositories were obtained surgically,thus the fertilized eggs were separated.Meanwhile,the spurious pregnant females were obtained synchronously by ligated male rats.Obtain sg RNA,Cas9 and Donor c RNA by in vitro transcription,mix them and microinject them into the obtained fertilized eggs.Healthy fertilized eggs were then selected and transplanted back into the pseudo-pregnant female.After the newborn mice were born,the toe of the newborn mice was clipped and identified by PCR,and transgenic positive mice were obtained.At the same time,the positive mice were cross-bred with wide type(WT)mice in the same background to establish the Mut-hHtrA1^L364P mice mice population.Transgenic homozygous mice were obtained and preserved by sperm freezing.Vascular smooth muscle cells(VSMCs)from homozygous transgenic mice were isolated and the expression of SM22 alpha was detected by cellular immunofluorescence technique to confirm the artificial VSMCs.On this basis,real-time fluorescence quantification polymerase chain reaction(RT-PCR),Western Blot,immunofluorescence technology and other methods were used to detect the expression and localization of HtrA1^L364P in transgenic homozygous mice VSMCs.Part ?:Phenotypic analysis and pathogenesis of Mut-hHtrA1^L364P miceFirstly,phenotypic analysis of Mut-hHtrA1^L364P mice was carried out:Observe its appearance and action state.Conduct behavioral analysis:including food maze and water maze;Pathological examination:Including routine hematoxylin-eosin staining(HE),Nissl staining,special staining of elastic fibers,specific antibodies:smooth muscle actin(SMA),actin(Actin),CD31,CD68 labeled immunohistochemical staining and the cell structure and subcellular structure were observed by electron microscopy.Secondly,the VSMCs of Mut-HtrA1^L364P mice were tested for cell proliferation,apoptosis and activity.The key factors of TGF-?signaling pathway(TGF-?,Smad2,Smad3,Smad4)were localized and qualitatively identified by immunohistochemistry.RT-PCR and Western Blot experiments were conducted to quantitatively detect the expression of genes and proteins in Mut-hHtrA1^L364P mice.Result:Part ? Results:Construction of Mut-hHtrA1^L364P mice:Relevant sequence information was retrieved and the location of each signal peptide was analyzed.The best sequence of sg RNA was confirmed by T7E1 digestion:TCGGGGACCGGCCGCTCGGC.This sequence has the highest intracellular activity.At the same time,the sequence of Donor vector was verified by sequencing.Fifteen,six and thirteen newborn mice were obtained by three batches of microinjection.Six F0-positive female mice were identified by PCR.After hybridization with background WT mice,they continued to breed and feed.Homozygous F2 mice were identified by gene sequencing,and Mut-hHtrA1^L364P mice population was established.Finally,the sperm was successfully frozen.Identification of Mut-hHtrA1^L364P mice:in the VSMCs ofHtrA1^L364P mice successfully isolated fromHtrA1^L364P mice,RT-PCR Western Blot,immunofluorescence and other experimental results showed that Mut-HtrA1^L364P mice only expressed human mutant HtrA1^L364P,but did not express their own background wild-type HtrA1,which was mainly expressed in the cytoplasm of VSMCs.Part ? resultsPhenotypic analysis of Mut-hHtrA1^L364P mice:compared with WT mice of the same age,Mut-hHtrA1^L364P mice showed obvious sparse hair,more white hair and clumsy and slow movement.The results of the food maze experiment showed that the time of finding food for Mut-hHtrA1^L364P mice and WT mice was 144.93±54.63 seconds and60.93±53.60 seconds,respectively,with a significant difference between the two groups(p<0.001).The number of errors in finding food in the two groups was1.93±1.16 seconds and 1.53±0.83 seconds,respectively,with no significant difference(p>0.05).The water maze experiment data showed that the first time ofHtrA1^L364P mice and WT mice to contact the escape table within 120 seconds was44.02±25.05 seconds,11.57±8.50 seconds,respectively,and the number of times of shuttle in the escape table quadrant was 1.75±0.97 seconds and 5.83±2.25 seconds,respectively,with extremely significant differences(p<0.001).Pathological test results showed that there was no significant difference in the structure,number and distribution of neurons betweenHtrA1^L364P mice and WT mice.Staining with HE,elastic fiber,SMA,Actin,CD31 and CD68 comprehensively showed that some small arteries in the brain ofHtrA1^L364P mice lost normal structure,with structural disorder,stenosis of lumen and degeneration of elastic fiber.Electron microscopy showed that the cortex and hippocampus of Mut-hHtrA1^L364P mice aging neurons more than WT mice,mitochondria,golgi apparatus and endoplasmic reticulum and other organelles appear abnormal morphology,synapses,capillaries,venules,and small artery wall thickening and luminal stenosis abnormal phenomenon,and there are more fat brown pigment deposition,appears more autophagy body at the same time.Pathogenic mechanism ofHtrA1^L364P mice:MTT and CFSE results showed that compared with WT mice,the viability of VSMCs derived fromHtrA1^L364Pmice was significantly decreased(p<0.01)and the proliferation rate was significantly slowed down(p<0.001).Annexin V-FITC/PI double staining and Tunel method showed that the apoptotic rate(FITC labeling rate)of primary VSMCs ofHtrA1^L364P was significantly higher than that of VSMCs of WT mice(p<0.001).RT-PCR results showed that compared with WT mice,the expression of TGF-?was significantly increased inHtrA1^L364P mice(p<0.001),the expression of Smad2and Smad3 was significantly increased(p<0.01),and the expression of Smad4 was not significantly different between the two groups(p>0.05).Western Blot test showed that the expression levels of TGF-?and Smad2 inHtrA1^L364P mice were significantly higher than those in WT mice(p<0.01),while the expression levels of Smad3 were significantly higher(p<0.05),and there was no significant difference in the expression levels of Smad4 between the two groups(p>0.05).Conclusion:1.Mut-hHtrA1^L364P mice was successfully constructed.The mice only expressed human mutant HtrA1^L364P,but not its own background wild-type HtrA1.2.Mut-hHtrA1^L364P mice can well simulate the pathogenesis of CARASIL in behavioral and pathological aspects,which can be used as a model to study the disease of CARASIL.3.In Mut-hHtrA1^L364P mice,it was found that TGF-?/Smad signal pathway was abnormally up regulated,which was involved in the pathogenesis of CARASIL disease.