Construction,Propagation And Identification Of TauT Knockout Rats Mediated By CRISPR/Cas9 System

Objective Taurine(Taurine)is a sulfur-containing amino acid that regulates cell osmotic pressure,prevents intracellular calcium overload,reduces free radical production,improves cell membrane permeability,and prevents cell swelling and deformation [1,2].An important trophic factor during the development of the central nervous system of infants,which enhances synaptic transmission and relieves neuroprotective effects such as stroke,epilepsy,and neurodegenerative diseases [3,4].Tau is mainly transported into cells by a sucrose carrier family 6 member 6,Slc6a6: SEQ ID NO: NC-005103.4;taurine transporter,Tau T.To investigate the effects of taurine deficiency on neurological diseases,this study used CRISPR/Cas9 technology to construct and propagate Tau T knockout rats(Tau T-/-)by Slc6a6 m RNA and Tau T protein in the Tau T knockout rat.The expression and physiological and biochemical indicators were tested to provide a reference for the health evaluation and pharmacological effects of the Tau T knockout rat model;and to provide stable rats for the study of the effects of taurine deficiency on nervous system diseases.model.Methods 1.Construction and identification of Tau T heterozygous(Tau T+/-)first constructed rat: According to the 5th exon of Slc6a6 gene,a specific plasmid expression vector was constructed by CRISPR/Cas9 technology,through amplification,screening,enzymatic digestion,Extraction,purification,in vitro transcription,synthesis of sg RNA and Cas9 m RNA,respectively,through microinjection,injection of fertilized eggs,cultured in vitro,and then transferred into the surrogate mother.Genotype identification and sequencing analysis were performed on the first-established rats,and the F0 generation Tau T+/-first-established rats were screened(This part of the experiment was commissioned by the Institute of Medical Laboratory Animals of the Chinese Academy of Medical Sciences).2.Reproduction and identification of Tau T+/-rats: F0 generation Tau T+/-rats were co-caged with wild-type SD rats to obtain F1 generation rats,which were cut toe,extracted DNA,identified,and screened for F1 generation Tau T+/-Rats,and continue to mating with wild-type SD rats,amplifying the reproductive system,and thenscreening out F2 generation Tau T+/-rats,allowing Tau T+/-rats to reproduce spontaneously,and obtaining F3 generation Tau T-/-(Homozygous rat),Tau T+/+(negative rat)Tau T+/-(heterozygous rat).F3 rats were selected for the following experiments.3.Expression of Slc6a6 gene and Tau T protein in Tau T-/-rat brain tissue: Total RNA and protein in rat brain tissues were extracted from Tau T-/-and Tau T+/+,and the expression of Slc6a6 m RNA was detected by Real-time PCR.Experimental and immunohistochemical methods were used to detect the expression of Tau T protein.4.Expression of Slc6a6 gene in other organs of Tau T-/-rats: The expression of Slc6a6 m RNA in heart,liver,kidney and skeletal muscle of Tau T-/-and Tau T+/+ rats was detected by Real-time PCR.5.Tau T-/-rat physiological and biochemical indicators: observe the body weight changes of F3 generation Tau T-/-rats and Tau T+/+ rats from 1 to 6 months old,and detect 16-20 weeks old Tau T-/-,Tau T+/-and physiological indexes such as body length,tail length,body weight and organ weight of Tau T+/+,comparing organ weight,organ coefficient,and dirty brain ratio;taking blood from rats and detecting with automatic blood cell analyzer 30 blood routine indicators;23 blood biochemical indicators were analyzed by automatic biochemical analyzer.Results 1.Construction and identification results of Tau T+/-rat first mouse: Two F0 generation Tau T+/-rats were constructed for the first time,and the heterozygosity rate was 18.2%.Electrophoretic identification: Electrophoresis results of Tau T+/-rats showed two electrophoresis bands(702 bp,312 bp).It was confirmed by sequencing that a band in the Tau T+/-rat electrophoresis result lacked the exon 5 of slc6a6.2.Tau T+/-rat breeding results: F0 generation and wild-type SD rats were mated to obtain F1 generations : 4 Tau T+/-and 5 Tau T+/+;F2 generations had 41 Tau T+/-,32 Tau T+/+;F3 generations contained 29 Tau T-/-,69 Tau T+/-,34 Tau T+/+,F3 generation homozygosity is about 21.97%.During the breeding period,two Tau T-/-rats were found to be characterized by slow acting,and the overall mortality of F0-F3 rats was 6.22%.3.Identification of Tau T-/-rats: The Tau T-/-group rats contained a strip of 312 bp.TheTau T+/-group consisted of two electrophoresis bands(702 bp,312 bp);the Tau T+/+ group contained one band(702 bp),and the sequencing confirmed the F3 generation Tau T+/-,Tau T-/-two genotypes.The sequence deleted by the rat is identical to the deletion sequence of the F0 generation.4.Expression of Slc6a6 gene in Tau T-/-rat brain tissue: Compared with Tau T+/+ rats,the expression of Slc6a6 m RNA in Tau T-/-rat brain was hardly expressed;the expression of Tau T protein in Tau T-/-rat brain was significant.reduce.5.Expression of Slc6a6 gene in other organs of Tau T-/-rats: almost no expression of Slc6a6 m RNA in the heart,liver,kidney and skeletal muscle of Tau T-/-group rats,and rats in Tau T-/-group The expression of Slc6a6 m RNA in the organs was significantly lower than that in the Tau T+/+ group.6,Tau T-/-rat physiological and biochemical indicators detection:(1)Physiological indicators: The weight of kidney and other organs in the Tau T-/-group at 16-20 weeks old was significantly lower than that in the Tau T+/+ and Tau T+/-groups;the length and tail length of the three genotype rats were not significant.difference.The organ coefficient of the heart of the female Tau T-/-group was significantly higher than that of the Tau T+/+ group,and the organ coefficient of the liver of the female Tau T+/-and Tau T-/-groups was significantly lower than that of the Tau T+/+ group.The cerebral coefficient of the heart and lung of the female Tau T-/-group was significantly higher than that of the Tau T+/+ group,and the cerebral brain coefficient of the male Tau T-/-group kidney was significantly lower than that of the Tau T+/-group.(2)Blood routine indicators: Compared with the Tau T+/+ group,the percentage of eosinophils in the Tau T-/-group was lower,the percentage of basophils and the hematocrit were higher.(3)Blood biochemical indicators: Compared with the Tau T+/+ group,the Tau T-/-group had lower albumin and higher alkaline phosphatase.Conclusions 1.This study used the CRISPR/Cas9 technology to knock out the exon 5 of the Slc6a6 gene in SD rats,and established the Tau T+/-rat model for the first time.2.The homozygous Tau T-/-rat model was successfully established by breeding andidentification,and the Tau T-/-rat did not have embryonic lethality.The deletion gene can be stably inherited: the DNA deletion sequence of the F3 generation Tau T-/-group is consistent with the F0 generation;the expression of Slc6a6 m RNA in the brain,heart,liver,kidney and skeletal muscle of the F3 generation Tau T-/-group is significantly reduced,and the brain The expression of Tau T protein was significantly reduced in tissues.3.The physiological and biochemical tests of Tau T-/-rats showed that the lack of Tau T affected the content of physiological and biochemical indicators such as albumin and alkaline phosphatase in rats;the comparison of body weight,organ coefficient and visceral coefficient showed Tau T.The lack of weight results in a decrease in body weight in rats and affects the growth and development of heart,liver and kidney in female rats to varying degrees.4.The results of this experiment can provide reference for the health assessment and drug toxicology experiments of Tau T-/-rats;the establishment of Tau T-/-rat model is also a late study of taurine deficiency for nervous system diseases.The effect is provided by a stable rat model.