Establishment Of Zebrafish Model With DHFR Gene Knockin And Experimental Researches On Its Cardiac Effects

Background:Congenital heart disease(CHD)is a morphological and structural abnormality of the heart and blood vessels in the cardiovascular system during embryonic development,resulting in hemodynamic changes.The clinical prognosis is poor,and severe types of CHD can cause miscarriage,stillbirth,stillbirth,neonatal death,and limited work capacity in children,adolescents,and adults.CHD ranks first in the incidence of perinatal congenital disabilities and can seriously affect people’s health.Therefore,it is increasingly important to determine the various pathogenic mechanisms of CHD.In the previous work,the incidence of copy number variants in PA patients and the role of copy number variants in the genetic pathogenesis of CHD were investigated using collected samples of PA patients and their parents.DHFR gene encodes dihydrofolate reductase,a key enzyme in the folate metabolic cycle that catalyzes the synthesis of dihydrofolate and tetrahydrofolate.Folic acid is an extremely important nutrient in heart development.Thus,the dhfr gene has a vital role in cardiac development.Methods:By CRISPR/Cas9 gene-editing technology,suitable knock-in sites were designed on the zebrafish dhfr gene,and in vitro synthesized sg RNA and Cas9 m RNA with specificity were microinjected into the zebrafish fertilized eggs.Then the activity of sg RNA was verified to confirm its effectiveness on the selected sites.The F0 generation zebrafish were raised to sexual maturity,and the F1 generation was obtained after free mating,which was screened for zebrafish mutants.By free mating,stable dhfr knock-in strains of zebrafish were obtained.To further analyze the effect of dhfr knock-in on the heart of zebrafish,on the one hand,the pure zebrafish of the F3 generation were crossed with the pure zebrafish of Tg G fluorescent transgenic strain to obtain the hybrid fluorescent strain zebrafish.Then free mating was performed,and the juvenile zebrafish were photographed and recorded under a fluorescence microscope for five days after fertilization to observe the heart alteration.On the other hand,sections of adult fish hearts were stained by the hematoxylin-eosin staining method to observe the myocardial tissue alterations.Results:By free mating,a stable dhfr gene knock-in strain of zebrafish was obtained.A total of 10 F1 generation zebrafish with dhfr gene knock-in were screened.The above experimental results showed that dhfr knock-in had significant effects on zebrafish heart development,appearing as pericardial edema,linear heart malformation,loose myocardial tissue,and tail curvature phenotypes.The expression of dhfr gene in zebrafish heart was analyzed by protein immunoblotting technique,and the experimental results suggested that dhfr was expressed in zebrafish heart tissues.The expression of Dhfr protein was elevated in dhfr knock-in zebrafish heart,which proved that dhfr gene knock-in was successful.Conclusions:DHFR gene plays an essential regulatory role in heart development,and copy number variants of this gene may constitute a novel pathogenic mechanism in CHD.