Knockout of ISCA 1 causes early embryonic death in rats

摘要

Background:I ron-sulfur cluster assembly 1(I SCA1)is an iron-sulfur(Fe/S)carrier protein that accepts Fe/S from a scaffold protein and transfers it to target proteins including the mitochondrial Fe/S containing proteins.ISCA 1 is also the newly identified causal gene for multiple mitochondrial dysfunctions syndrome(MMDS).However,our knowledge about the physiological function of ISCA 1 in vivo is currently limited.In this study,we generated an ISCA 1 knockout rat line and analyzed the embryo development.Methods:I SCA 1 knockout rats were generated by replacing the exon1 of I SCA 1 gene with the mC herry-Cre fusion gene using CRISPR-Cas9 technology.The ISCA 1 expression pattern was analyzed by fluorescence imaging using ISCA 1 promotor driven Cre and mC herry expression.The embryonic morphology was examinated by microscope and mitochondrial proteins were tested by Western blot.Results:A n ISCA 1 knockout rat line was obtained,which expressed m Ch erry-Cre fusion protein.Both of the fluorescence images from mC herry and Cre induced mC herry in a reporter rat strain,showing that ISCA 1 expressed in most of the tissues in rats.The ISCA 1 knockout resulted in abnormal development at 8.5 days,with a significant decrease of NDUFA 9 protein and an increase of aconitase 2(ACO 2)in rat embryos.Conclusion:D eletion of I SCA 1 induced early death in rats.I SCA1 affected the expression of key proteins in the mitochondrial respiratory chain complex,suggesting that ISCA 1 has an important influence on the respiratory complex and energy metabolism.