Article Figures & Data
Figures
- Fig. 1.
Schematic representation of SULT4A1 mutations generated by CRISPR-Cas9. (A) Diagram of the Δ28 mutant SULT4A1 mouse line. CRISPR-Cas9 technology induced a 28-bp deletion within exon 1 that resulted in a frameshift mutation and premature stop codon at AA 62. (B) Diagram of the Δ12 mutant SULT4A1 mouse line. Gene editing induced a 12-bp in-frame deletion of the 4 AA immediately preceding the active site His. For both lines, SULT4A1 female founders were used to establish breeding colonies.
- Fig. 2.
Characterization of SULT4A1 protein expression in Δ28 and Δ12 mutant mouse brains and subcellular localization of WT SULT4A1. WT, heterozygous, and homozygous Δ28 and Δ12 25-day-old mice were euthanized and whole brains were harvested. (A) WT, heterozygous (HET), and homozygous (MUT) Δ28 and Δ12 mice were probed for SULT4A1 protein expression. Human brain cytosol was used as a positive control (CTRL) for SULT4A1. For both strains, WT and heterozygotes showed positive staining for SULT4A1; however, Δ28 and Δ12 homozygotes were negative for SULT4A1. (B) Subcellular fractionation was used to generate nuclear (Nuc), mitochondrial (Mito), cytosolic (Cyto) and microsomal (Micro) fractions from WT adult mouse brain homogenates. Aliquots of each fraction (50 μg) were subjected to SDS-PAGE and immunoblotted for SULT4A1. To determine the purity of subcellular fractions, marker proteins specific to each fraction were probed. Histone H3 was used to probe nuclear fraction purity, mitochondrial import receptor subunit TOM20 homolog (TOM20) was used as a marker protein for the mitochondrial fraction, GAPDH was used as a marker for the cytosolic fraction, and Na+/K+ ATPase was used to determine the microsomal fraction purity. SULT4A1 protein was detected in the mitochondrial, cytosolic, and microsomal fractions, and marker proteins demonstrated pure subcellular fractions.
- Fig. 3.
SULT4A1 expression in embryo-derived mouse cortical neurons by confocal microscopy. Cortical neurons were harvested from embryonic day-15 mice and grown on cover slips for an additional 12 days prior to immunocytochemistry. Cortical neurons were probed for SULT4A1 expression using a rabbit polyclonal IgG at a 1:5000 dilution. Nuclei were labeled with Hoechst (1:20,000) with primary cortical neurons being positive (green) for SULT4A1 protein in both the soma and neurites, but not in the nuclei (blue).
Additional Files
Data Supplement
- Supplemental Data -
Supplemental Methods
Supplemental Figure 1 - Electropherogram tracings confirm mutational status of SULT4A1 was
conserved in offspring of Δ28 and Δ12 founders - Movie 1 -
Δ28 and Δ12 homozygous mutant mice display severe neurological deficits
- Movie 2
- Supplemental Data -
In this issue
SULT4A1 Mutant Mouse Models
