Knockout of ISCA1 causes early embryonic death in rats

Abstract

Background: Iron-sulfur cluster assembly 1 (ISCA1) 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. ISCA1 is also the newly identified causal gene for multiple mitochondrial dysfunctions syndrome (MMDS). However, our knowledge about the physiological function of ISCA1 in vivo is currently limited. In this study, we generated an ISCA1 knockout rat line and analyzed the embryo development.

Methods: ISCA1 knockout rats were generated by replacing the exon1 of ISCA1 gene with the mCherry-Cre fusion gene using CRISPR-Cas9 technology. The ISCA1 expression pattern was analyzed by fluorescence imaging using ISCA1 promotor driven Cre and mCherry expression. The embryonic morphology was examinated by microscope and mitochondrial proteins were tested by Western blot.

Results: An ISCA1 knockout rat line was obtained, which expressed mCherry-Cre fusion protein. Both of the fluorescence images from mCherry and Cre induced mCherry in a reporter rat strain, showing that ISCA1 expressed in most of the tissues in rats. The ISCA1 knockout resulted in abnormal development at 8.5 days, with a significant decrease of NDUFA9 protein and an increase of aconitase 2 (ACO2) in rat embryos.

Conclusion: Deletion of ISCA1 induced early death in rats. ISCA1 affected the expression of key proteins in the mitochondrial respiratory chain complex, suggesting that ISCA1 has an important influence on the respiratory complex and energy metabolism.

Keywords: ISCA1; embryonic development; energy metabolism.

Figure 1

The establishment of ISCA1 knockout rats. A, The mCherry‐Cre cassette was knocked in at the ISCA1 locus, replacing the exon1 of ISCA1 gene. B, The mutated rats were genotyped by PCR. C, The heterozygous ISCA1‐mCherry‐Cre rats (ISCA1 mCherry‐Cre ^−/+) were crossed with the Rosa26‐imCherry reporter rats. A strong expression of mCherry protein was induced by Cre (right) compared with a WT rat (left). The mCherry fluorescence images were captured by In‐Vivo Imaging Systems

Figure 2

The expression pattern of ISCA1. A, The tissues of ISCA1‐Cre‐mCherry ^−/+ rats were sampled and frozen sections of the tissues were examinated using the NanoZoomer family of Digital Pathology Systems (magnification× 40, scale bar = 100 μm). B, The expression of ISCA1 in the tissues was further analyzed by RT‐PCR and C, quantified by gray scanning and normalized to GADPH (n = 6)

Figure 3

Observation of the mCherry‐Cre ^−/− embryos. A, The male and female heterozygous rats (ISCA1‐mCherry‐Cre ^−/+) were mated with each other and the numbers of WT, heterozygote and homozygote litters (ISCA1‐mCherry‐Cre ^−/−) were counted. The embryonic morphology was observed and genotyped at B, 2.5 d, and C, 8.5 d

Figure 4

The expression of NDUFA9 and ACO2. A, The total embryo lysates from WT and homozygous embryos (ISCA1‐mCherry‐Cre ^−/−) were separated on SDS‐PAGE and the expression levels of NDUFA9 and ACO2 detected by Western blot. The levels of B, NDUFA9 protein and C, ACO2 protein were quantified by gray scanning and normalized to TOMM20 (n = 3, *P < 0.05)