Establishment of a Dihydrofolate Reductase Gene Knock-In Zebrafish Strain to Aid Preliminary Analysis of Congenital Heart Disease Mechanisms

Abstract

Background: The dihydrofolate reductase (DHFR) gene is imperative in development, therefore it is essential to explore its effects on heart development. Thus, here a dhfr zebrafish knock-in (KI) strain was constructed.Methods: CRISPR/Cas9 technology was used to establish the dhfr KI zebrafish strain. This strain was hybridized with TgG fluorescent strain zebrafish to observe the phenotypes of heart shape, size, and circularization direction. Wild-type (WT) and KI zebrafish were then dissected and histologically stained to observe pathological changes. Western blot analysis was used to verify the increased expressions of zebrafish genes after KI. Hybridization experiments were used to confirm the presence of abnormal gonadal dysplasia.Results: The zebrafish dhfr KI strain was successfully constructed through CRISPR/Cas9 technology. At 6 days post fertilization (dpf), microscopic examinations of KI (homozygous) specimens revealed pericardial effusions, heart compressions, and curled tails. Compared with WT, the Hematoxylin and Eosin (H&E) tissue sections of KI-homozygous zebrafish showed defects such as reduced atria and ventricles. Western blot analysis indicated that the expression of the DHFR protein increased in both heterozygotes and homozygotes of dhfr KI zebrafish. Hybridization experiments revealed that dhfr KI may affect gonadal function.Conclusion: The DHFR gene plays an important regulatory role in the process of heart development, and copy number variations (CNVs) of this gene may constitute a new pathogenic mechanism of congenital heart disease (CHD).

Genome-wide analysis of methylation in rat fetal heart under hyperglycemia by methylation-dependent restriction site–associated DNA sequencing

“…The DNA methylation status of the upstream region is critical because of its role in regulating gene expression [ 20 ]. Two genes with abnormal CCGG methylation in the upstream region, Smad3 [ 21 ] and Dhfr [ 22 ], were selected from the results of Methylation-RAD sequencing because of their previously established roles in CHD. qRT-PCR showed that the expression of smad3 mRNA was decreased ( Fig 5A ) and the expression of dhfr mRNA was increased in the diabetic group ( Fig 5B ).…”

Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping

“…Both Cas9 protein and mRNA have successfully been used in the past to generate zebrafish KI lines 14 , 24 , 27 , 28 . Despite similar somatic editing efficiencies, higher rates of germline transmission have been observed for protein compared to mRNA 12 .…”

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species

“…Another approach to modeling is to simulate the symptoms of the target disease through mutations in genes with known function. Individuals and lines have been created that exhibit analogs of human somatic diseases such as cataract [ 49 , 50 ], myopia [ 51 ], exfoliative syndrome [ 52 ], retinal dysfunction [ 53 , 54 ], congenital heart defect [ 55 ], cardiac hypertrophy [ 56 ], dilated cardiomyopathy [ 57 ], arrhythmia [ 58 ], autoinflammatory syndrome [ 59 ], metabolic syndrome [ 60 ], diabetes and obesity [ 61 ], tuberculosis [ 62 ], thrombocytopenia [ 63 ], pediatric intestinal pseudoobstruction [ 64 ], pediatric cirrhosis [ 65 ], congenital hypothyroidism [ 66 ], fatty or alcoholic hepatosis [ 67 ], and scoliosis [ 68 , 69 ]. Genome editing has been used to model human tumors such as liver cancer [ 70 ], paraganglioma [ 71 ], skin melanoma [ 72 ], and epithelioid sarcoma [ 73 ].…”