Establishment Of Rat Model Of Abbb1 Knockout And Humanized Rat Model And Establishment Of AhR Humanized Rat Model And Acute Toxicity Analysis Of TCDD To AhR Humanized Rats

Objective P-gp is a protein which coded by Abcbl,it is related to the drug transport in the blood-brain-barrier and other tissues, and it is closely connected with the multidrug resistance of tumor cells. There have not been suitable animal models to evaluate the ability of the drug to go through the blood brain barrier and enter the tumor cell. The goal of our study is to knock out the Abcbl gene of rat,and establish the Abcbl humanized rat model based on the Abcbl knock out rat. To provide a animal model closed to human which can evaluate the Abcb1 related drug.Methods The BAC-Abcb1 was constructed by bacterial artificial chromosome, the sgRNA was designed and constructed by CRISPR/Cas9.These two kinds of transgenic rats were generated by microinjection and were all maintained on SD genetic background. The Abcbl humanized rats were generated by hybrid breeding. The animal model were identified and analyzed by PCR, sequencing, RT-PCR and real-time PCR.Results Establishing a rat model expressing human Abcb1 stably by transfer the 153kb BAC containing human Abcb1 promoter and cDNA into rat genome, and establishing the Abcbl knock out rat at the same time. Establishing the Abcbl humanized model by crossing these two strains together. The Abcb1 humanized model express not only the human Abcbl gene but also has similar expression pattern as human.Conclusions The Abcbl knock out rat and the Abcbl humanized rat were successfully established, and this model which close to human on Abcbl.can be used to evaluate the metabolism of related drugs.Objective AhR is the key participant in the acute toxicities and carcinogenicity observed following exposure to TCDD and a variety of other polycyclic, polyhalogenated hydrocarbons, and AhR plays a critical role in the development and function of multiple tissues.The sensitivity of different species to TCDD are different and there has not been suitable animal models to evaluate the TCDD toxicology on human. The goal of our study is to knock out the AhR gene of rat, and establish the AhR knock out rat to explore the function of AhR gene, and establish the AhR humanized rat model based on the AhR knock out rat. To provide an animal model closed to human which can evaluate the AhR related drugs and poisons.Methods The BAC-AhR was constructed by bacterial artificial chromosome, the sgRNA was designed and constructed by CRISPR/Cas9.These two kinds of transgenic rats were generated by microinjection and were all maintained on SD genetic background. The AhR humanized rats were generated by hybrid breeding. The animal models were analyzed by PCR, Sequecing, RT-PCR, Real-time PCR and Western blot. Wild type rats, AhR knockout rats and AhR humanized were given single intraperitoneal injection of TCDD once respectively, the blood biochemistry,organ coefficient and pathological changes of major organs were observed after administration to evaluate the sensitivity of three rat strains to TCDD,and speculating the human’s sensitivity to TCDD.Results Establishing a rat model expressing human AhR stably by transfer the 162kb BAC containing human AhR rat genome, and establishing the AhR knock out rat at the same time. Establishing the AhR humanized model by crossing these two strains together. The expression pattern of AhR in AhR humanized rat is different from the wild type rat. The AhR humanized model express not only the human AhR gene but also has similar expression pattern as human. The sensitivity of The AhR knock out rats and the AhR humanized rats to TCDD are less than the wild-type rats, AhR may play a critical role in heart,liver,lungs and thymus development process.Conclusions The AhR knock out rat and the AhR humanized rat were successfully established, and the AhR knock out can be used to evaluate the AhR function, and the AhR humanized model is close to human concerning about the drug and poisons metabolism related to AhR.