Knockout of ISCA1 causes early embryonic death in rats

Abstract

Background Iron‐sulfur cluster assembly 1 ( ISCA 1) 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 ISCA 1 knockout rats were generated by replacing the exon1 of ISCA 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 An ISCA 1 knockout rat line was obtained, which expressed mC herry‐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 Deletion of ISCA 1 induced early death in rats. ISCA 1 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.

Conditional knockdown of integrin beta-3 reveals its involvement in osteolytic and soft tissue lesions of breast cancer skeletal metastasis

“…ISCA1 relocates Fe/S from a scaffold protein to the mitochondrial proteins from the respiratory complex, which is necessary for their stability. It has been reported that ISCA1 knockout led to impairment of the electron transport chain and energy metabolism (Yang et al 2019). Here, following the conditional ITGB3 knockdown, ISCA1 was persistently downregulated, which might be a hint for impeded cellular energy metabolism.…”

“…The effects observed following ITGB3 knockdown in vitro and in vivo can be explained by downregulation of specific genes, which have roles in angiogenesis (NPTN, RRM2) (Rodriguez-Pinto et al 2009;Chen et al 2019), tumor growth (NPTN) (Rodriguez-Pinto et al 2009), energy metabolism (ISCA1) (Yang et al 2019), cytokinesis (SEPT11) (Hanai et al 2004), migration (RRM2, STX6) (Chen et al 2019;Zhu et al 2016), cell proliferation, invasiveness, senescence, tumorigenesis (RRM2) (Chen et al 2019) and vesicle trafficking (SEPT11, STX6) (Hanai et al 2004;Zhu et al 2016). It is known that the high expression of neuroplastin (NPTN) in breast carcinomas with distant metastasis is related to a significant increase in tumor growth and angiogenesis (Rodriguez-Pinto et al 2009).…”

“…In our study, the NPTN expression was persistently down-regulated in MDA-MB-231 breast cancer cells upon ITGB3 knockdown. Iron-sulfur cluster assembly 1 (ISCA1) is a mitochondrial protein, associated with the biogenesis of iron-sulfur clusters (Cozar-Castellano et al 2004), which are essential cofactors for cellular processes like mitochondrial respiration and DNA metabolism (Yang et al 2019). ISCA1 relocates Fe/S from a scaffold protein to the mitochondrial proteins from the respiratory complex, which is necessary for their stability.…”

ISCA1 Deficiency Induces Iron Metabolism Disorder and Myocardial Oncosis in Rat

“…To investigate the effects of ISCA1 on organ development, we first established ISCA1 knockout rats by replacing the exon 1 of ISCA1 gene with the mCherry-Cre fusion gene using CRISPR-Cas9 technology in our lab as previously reported [48]. We found that knockout homozygous rats (Isca1 mCherry-Cre -/-) cannot be available in the pups born, which arrested fetal development at E8.5 [48].…”

“…To investigate the effects of ISCA1 on organ development, we first established ISCA1 knockout rats by replacing the exon 1 of ISCA1 gene with the mCherry-Cre fusion gene using CRISPR-Cas9 technology in our lab as previously reported [48]. We found that knockout homozygous rats (Isca1 mCherry-Cre -/-) cannot be available in the pups born, which arrested fetal development at E8.5 [48]. Thus, to further explore how ISCA1 shapes heart morphology and function in vivo, we first established Isca1 conditional knockout (Isca1 cKO) rat in this study (Figure 2A), and selectively eliminated ISCA1 from the α-MHC rat genome by crossing Isca1 cKO rats with the α-MHC-Cre rats (Figure 2B).…”