Study On Zebrafish Rsph3 Gene In Early Development And Preparation Of Zebrafish Prdx Mutants

Gene knockout technology is an efficient method to study reverse genetics.The knockout tools like CRISPR/Cas9 and TALENs have been proved effective.Zebrafish is an animal model for analysizing functional genomics in studying the mechanism of human diseases.Primary ciliary dyskinesia is a heterogeneous disease which is caused by the developmental dysfunction of cellular cilia structure.Clinical manifestations include neonatal respiratory distress syndrome,chronic respiratory infections and bronchiectasis,blindness,deafness and visceral inversion body on the right side of the heartas well as obesity,diabetes and so on..Radial spokes protein 3(RRPH3)is one of the 20 kinds of protein in radial spokes complex and is necessary for axonemal slip.We used TALENs technology to delete rsph3 gene in zebrafish.We got three mutants of rsph3 heterzygote(A8/10/15 bp)and two homozygote mutants of rsph3(?8/15 bp),which generated truncated proteins.In situ hybridization,our results showed rsph3 widely expressed in zebrafish embryonic development and maternal stages.When the rsph3-/-mutants are reciprocal crossed,the cleavage rate of fertilized egg is 48%.In the survived animals,we found the liver and hear located right side,the heart tube is not bending,and displayed the destroyed left-right asymmetry axis.Dand5,the marker gene of kupffer's vesicle,expressed normal on mutant and wild type and did not completely surround "C" shape in 6 somite period.The speculation about the destruction of the symmetry axis may not be due to structural abnormalities of KV,which caused by abnormal ciliary structure and function.Zebrafish rsp3-/-mutant can be used as an animal model of cilium to understand human disease.Peroxiredoxins is an enzyme to play important role in the process of eliminating reactive oxygen species in cells.Using the CRISPR/Cas9 technology to knock out the zebrafish prdx family,we got three mutants of prdxl,prdx5,prdx6.Among these mutants,we got prdx1,prdx6 F0 generation,prdx5 heterzygote(A2,A5,A8)and the mutant of prdx5 homozygote(?5)which generated truncated proteins.In situ hybridization,our results showed that the zebrafish prdxl,prdx5,prdx6 were maternal expressed and existed widely in the early embryo.RT-PCR results showed prdxl,prdx5,prdx6 in adult ovary and testis have a high expression quantity.prdx5 mostly expressed in testis.We found that prdx5+/-embryonic zebrafish was more vulnerable to 15 mmol/L H2O2 treatment.