PMC

(A) Cotransfection of Alms1a siRNA-treated cells with a 5′ Alms1 cDNA construct rescues primary cilia formation in mIMCD3 cells.
(B) Real-time PCR analysis of Alms1a siRNA and N-terminal Alms1-transfected cells. Upper panel: over-expression of the 5′ cDNA does not affect knockdown of endogenous Alms1 mRNA with Alms1a siRNA. Lower panel: knockdown of endogenous Alms1 mRNA does not affect overexpression of the 5′ cDNA. N, negative control siRNA; cDNA, 5′ Alms1 cDNA; 1a, Alms1a siRNA.
(C) Stable expression of Alms1 mRNA from the Alms1^{L2131X/L2131X} allele. Real-time PCR analysis of Alms1 gene expression in an Alms1^{L2131X/L2131X} mouse and a wild-type littermate control.
(D) The N-terminal mouse Alms1 antibody detects Alms1 mutant protein at the ciliary basal body in primary kidney cells from the Alms1^{L2131X/L2131X} mouse. Shown are low and high magnifications of the ciliated cells. Arrowheads point out the base of cilia.
(E) Normal appearance of primary cilia in primary fibroblasts (MEF) and primary kidney cells (PKC) from the Alms1^{L2131X/L2131X} mouse strain.
(F) Inhibition of cilia formation in Alms1a siRNA-treated Alms1^{L2131X/L2131X} primary fibroblasts. Scale bars, 10 μm.

Workflow of ALMS1 endogenous tagging in HEK293T cells using CRISPR/Cas9.A, schematic overview of the ALMS1 gene with 23 exons (blue) and the site for tag insertion at the C-terminal end of ALMS1 (upper panel). Enlarged the sgRNA targeting site (grey), PAM sequence (blue), and the stop codon (red) are depicted. Cas9 cuts (purple) three nucleotides upstream of the PAM sequence. B, simultaneous transfection of ALMS1 targeting sgRNA cloned into the CRISPR/Cas9 (pX459.2) vector, and the sfGFP sequence harboring repair construct into HEK293T cells was performed. PCR-amplified products of ALMS1 CT region of HEK293T single clones were sent in for sequencing, confirming sfGFP tag insertion at the CT end of ALMS1 (example shown in lower panel). Bold italic marked base pairs were wobbled to prevent repeated cutting after tag insertion. C, native ALMS1 localized at the basal body of cilia in HEK293T cells (G0 phase). ARL13B was used as a ciliary marker. ALMS1 is depicted in green, ARL13B in magenta, and DAPI in light blue. The scale bar measures 5 μm. D, endogenously sfGFP-tagged ALMS1 (ALMS1-sfGFP) in HEK293T single clones in the G0 phase. Cells were stained with ARL13B (magenta) as a ciliary marker and ALMS1 (magenta) as a basal body marker. DAPI is depicted in light blue. The scale bar measures 5 μm.

A) Zinc finger nucleases target 17 base pairs in exon 1 of the ALMS1 gene leading to a frameshift and causing a pre-mature stop codon in exon 2 (figure adapted from []). B) Representative Western blot for ALMS1 in kidney lysates from WT and ALMS1 KO rats, n = 3, * p < 0.05 vs. WT. Data is represented as mean ±SEM; analyzed by a two-tailed Student’s t-test.

Mutation in the ALMS1 gene.