Study On The Transgenic Rat Model Of Congenital Hypothyroidism Via CRISPR/cas9

Congenital hypothyroidism(CH) is a common endocrine metabolic disease. The patients are suffered from physical retardation and intelligent developmental disorders. Because of its high incidence and can lead to retardation of many organs of the body, so it is necessary to further study CH effect mechanism of normal development of organs. Due to ethical issues,human beings cannot continue research when make a diagnosist of CH, so we must depend on animal disease models.But because lack of CH rat model of gene knockout, so the researchers obtain the rat model of CH was induced by drugs, which obviously cannot truly simulate due to the impact caused by mutations of CH on rat body development, so by knocking out the regulation of thyroid gland development or thyroid hormone synthesis related the gene to construct CH rat model has become a necessary precondition to study CH.In our research, we generated Tshr-/- knockout rat model by CRISPR/cas9 gene-editing technology,The model can provides a basis for the mechanism of thyroid hormone in the future finally clear during organ development and pathological mechanism of CH.The main contents of research:1.Build construction and the RNA transcription in vitroThe pX330 gene cloning plasmid, namely via restriction endonuclease,connections and processes to expand training steps to obtain objective, such as plasmids. Purpose plasmid transfection of rat BRL cells after a successful knock efficiency verification. Select high efficiency purpose of plasmids in vitro transcription, get cas9 RNA and sgRNA.2.the Zygote microinjection and transplantationSD wild-type female mice ovarian stimulation to get one-celled fertilized eggs,use micro-injection platform to inject cas9 RNA and sgRNA, then select quality embryo to transpant to the clean SD leave-successfully pregnant female rats inside the fallopian tube, normal deliveries after the transplant.3.The generation rats genotypic identification and off-targeting analysisAfter 21 st day when the rats born, take Salvia to extract genomic DNA and todesign primers for gene sequencing analysis. Select 5 mismatch sites to off-targeting analysis.4.The phenotype and the Hormone analysisGene knockout rats continued conventional breeding, to long observation records phenotypes such as weight, in its 13 th week using enzyme-linked immunosorbent assay(Elisa) to test T3,T4,TSH level of the wild-type of control group and experimental group.Results and conclusions:we generated Tshr-/- knockout rat model by CRISPR/cas9 gene-editing technology first in the world.