Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis
- Author:
- No author available
- Journal Title:
- Acta Physiologica Sinica
- Issue:
- 3
- DOI:
- 10.13294/j.aps.2017.0026
- Key Word:
- tnnt2a;CRISPR/Cas9;斑马鱼;tnnt2a;CRISPR/Cas9;zebrafish
Abstract： Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber,and participates in heart excitation-contraction coupling process.Here,we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique,and performed phenotypic analysis of the identified tnnt2a mutants.We observed that there was no significant difference between heterozygous mutant and wild type zebrafish,and the homozygous mutants displayed significant malformations in heart,including cardiac arrest,atrium and ventricle enlargement,pericardium effusion,and the individuals usually died before 7 day post fertilization (dpf).We further analyzed the expression alternations of heart sarcomere genes (tnnt2a,actc1a,tpm4a,myl7,vmhc) at transcriptional level in tnnt2a-/-(A2) zebrafish by performing real time RT-PCR,and found that the RNA expression level of tnnt2a in tnnt2a-/-zebrafish decreased constantly at each time point of developmental stages,and actcla,tpm4a,myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages,in comparison with those of wild type zebrafish.Lastly,electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a-/-(A2) zebrafish,which was further confirmed by F-actin and Tpm4 immunofluorescence staining.The tnnt2a-/-zebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy,and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.
- [1]Kobayashi T,Solaro RJ.Calcium, thin filaments, and the integrative biology of cardiac contractility.[J].2005,67.
- [2]Sampath K, Gollapudi,Murali, Chandra.The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by α- and β-myosin heavy chain.[J].2016.
- [3]Tardiff,J.C.,Schwartz,S.D.,Manning,E.P..Molecular effects of familial hypertrophic cardiomyopathy-related mutations in the TNT1 domain of cTnT[J].2012,421(1).
- [4]J C, Moolman,V A, Corfield,B, Posen,etc.Sudden death due to troponin T mutations.[J].1997,29(3).
- [5]Noboru Fujino,Masami Shimizu,Hidekazu Ino,etc.A novel mutation Lys273Glu in the cardiac troponin T gene shows high degree of penetrance and transition from hypertrophic to dilated cardiomyopathy[J].2002,89(1).
- [6]Sehnert AJ,Huq A,Weinstein BM,etc.Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.[J].2002,31(1).
- [7]J F, Forissier,L, Carrier,H, Farza,etc.Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy.[J].1996,94.
- [8].CRISPR/Cas system and its role in phage-bacteria interactions. [Review][J].2010,64(1).
- [9]Jian-Ping, Jin.Evolution, Regulation, and Function of N-terminal Variable Region of Troponin T: Modulation of Muscle Contractility and Beyond.[J].2016.
- [10]Wright, Addison V.,Nunez, James K.,Doudna, Jennifer A..Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering[J].2016,164(1/2).
- [11]Bournele, Despina,Beis, Dimitris.Zebrafish models of cardiovascular disease[J].2016,21(6).
- [12]Reda, Sherif M.,Gollapudi, Sampath K.,Chandra, Murali.L71F mutation in rat cardiac troponin T augments crossbridge recruitment and detachment dynamics against alpha-myosin heavy chain, but not against beta-myosin heavy chain[J].2016,37(6).
- [13]Roberts,J.A.,Miguel-Escalada,I.,Slovik,K.J.,etc.Targeted transgene integration overcomes variability of position effects in zebrafish[J].2014,141(3).
- [14]Marraffini, Luciano A..CRISPR-Cas immunity in prokaryotes[J].2015,526(Oct.1 TN.7571).
- [15]Nakajima Taniguchi C,Fujio Y,Nagata S,etc.Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy.[J].1997,29(2).
- [16]Rosevear P,Kobayashi T,Solaro RJ.The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation.[J].2008,369(1).
- [17]Uversky, Vladimir N.,Kong, Min J.,Pinto, Jose R.,etc.Troponins, intrinsic disorder, and cardiomyopathy[J].2016,397(8).