Generation of Cluap1 knockout allele mice
The Cluap1 knockout allele (Cluap1
, Knockout Mouse Project Repository, Davis, CA; hereinafter referred to as Cluap1
) was generated using embryonic stem cells in which a β-galactosidase-neomycin resistance fusion cassette was inserted into intron 2 of Cluap1. The insertion site was confirmed by genomic PCR and sequence analysis. PCR primers for genotyping were designed based on the insertion site (sequences available upon request). The embryonic stem cells containing the targeted allele were on the C57BL/6 N background and were injected into albino C57BL/6 blastocysts (C57BL/6 J-Tyrc-2 J; JAX Laboratories) by the UAB Transgenic Mouse Facility using standard procedures. Chimeras were then crossed with albino C57BL/6 females, and germline transmission was confirmed by the coat color of the offspring and subsequent PCR genotyping. After obtaining no homozygous mutant offspring from heterozygous matings, timed pregnancies were established to isolate embryos at the indicated gestational time point with the morning of the vaginal plug being considered embryonic day 0.5 (E0.5). Embryos were genotyped from DNA isolated from yolk sac by PCR. Mice were provided standard laboratory chow and water ad libitum. All procedures and studies involving mice were approved by the UAB Institutional Animal Care and Use Committee in accordance with regulations at the University of Alabama at Birmingham.
Reverse transcription PCR analysis
RNA was isolated from Cluap1
, and Cluap1
E9.5 embryos with Trizol reagent according to the manufacturer’s protocol (15596–026, Life Technologies, Carlsbad, CA). Once extracted, RNA was used to synthesize cDNA using the Verso cDNA kit according to the manufacturer’s protocol (AB-1453, Thermo Scientific, Pittsburgh, PA). PCR analysis was then performed using the following primers (written 5′ to 3′), which flank the sequence between the first and last exons of the Cluap1
allele: GGACTCGAGACCATGTCT and GGACCCGGGAAGAAGTCA. The following primers were also used as a positive control to confirm the presence of actin in all samples: ATGGGTCAGAAGGACTCCTA and GGTGTAAAACGCAGCTCA. All results were confirmed by repeating the experiment in at least two additional animals.
Cluap1 antibody generation
Antisera against Cluap1 was generated in rabbits by using a 19-residue peptide (KPSRRIRKPEPLDESDNDF) starting at position 395 of the mouse protein according to the standard protocol established by Open Biosystems (Huntsville, AL, USA). Specificity of the antisera against Cluap1 was confirmed by Western blot analysis of protein extracts isolated from Cluap1
, and Cluap1
IMCD3 cells (ATCC, Manassas, VA) were maintained in DMEM: F12 medium supplemented with 10% FBS, 1.2 g/l of sodium bicarbonate, 0.5 mM sodium pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin. NIH3T3 cells were cultured in DMEM with 10% FBS containing 100 U/ml penicillin and 100 mg/ml streptomycin. Creation of 176-6C renal epithelial cells was derived by microdissection of the cortical collecting duct segments of the kidney as previously described by Croyle et al.. To induce cilia formation, cells were serum starved for 24 – 48 h prior to analysis. All cells were grown at 5% CO2/95% air at 37°C.
Embryonic day 9.5 embryos were isolated into ice-cold lysis buffer [137 mM NaCl, 20 mM Tris pH 8.0, 1% Triton X-100, 10% glycerol, and complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN)]. Embryos were disrupted by passage several times through a syringe attached to a 30.5-gauge needle. The lysates were incubated on ice for 30 min and vortexed every 5 min. Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Protein samples were resolved on a denaturing 10% Tris–HCl gel (Bio-Rad Laboratories, Hercules, CA) and transferred to an Immobilon-Psq transfer membrane (Millipore, Billerica, MA). Membranes were blocked in TBS-T (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) with 5% milk for 1 h and incubated with primary antibody diluted in TBS-T with 2% BSA for 16–24 h at 4°C. Membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in TBS-T with 1% milk for 1 h at room temperature. Secondary antibodies were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Waltham, MA), and bands were visualized using Blue Ultra Autorad Film (Bioexpress ISC). The following primary antibodies and dilutions were used: anti-actin (Sigma; rabbit polyclonal; 1:1,000) and anti-Cluap1 (1:1,000). The secondary antibody was HRP conjugated anti-rabbit (#31460) and was used at 1:5,000 (Pierce/Thermo Scientific, Waltham, MA).
Whole kidney and heart were extracted from Cluap1
mice at 8 weeks of age. Tissues were fixed overnight at 4°C in 4% PFA in PBS and subsequently washed in PBS. Tissues were then cryoprotected with 30% sucrose in PBS for 24 h and snap frozen in OCT freezing compound (Tissue-Tek, Torrance, CA). Ten-micron sections were cut with a Leica CM1900 cryostat, and sections were attached to Superfrost Plus microscope slides (12-550-15, Fisher Scientific, Pittsburgh, PA). Sections were postfixed in 4% PFA in PBS for 10 min, washed three times with lacZ wash buffer (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, in 100 mM sodium phosphate buffer, pH 7.3), and then incubated in X-gal staining solution (2 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg ml-1 X-Gal, in PBS) at 37°C overnight. Sections were then counterstained in Fast Red for 5 min. Similarly, for whole-mount analyses E9.5 embryos and lung tissue from 8-week-old mice were fixed in 4% PFA in PBS, washed three times with lacZ wash buffer, and then incubated in X-gal staining solution at 37°C overnight.
Embryos and cells grown on coverslips were fixed in 4% PFA and permeabilized with 0.3% Triton X-100 in PBS with 2% donkey serum, 0.02% sodium azide, and 10 mg/ml bovine serum albumin (BSA). Embryos were then cut to make 10-μm sections. Cells and embryos were labeled with the following antibodies: anti-acetylated α-tubulin, 1:1,000 (T-6793; Sigma-Aldrich, St. Louis, MO); anti-Arl13b, 1:1,000 (a gift from Dr. Tamara Caspary, Emory University); anti-Cluap1, 1:1,000 (generated as described above); and anti_ShhN, 1:1,000 (5E1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). All incubations and washes were carried out in PBS with 2% normal donkey serum, 0.02% sodium azide, and 10 mg/ml BSA. Primary antibody incubations were performed for 16–24 h at 4°C, and secondary antibody incubations were performed for 1 h at room temperature. Secondary antibodies included Alexa Fluor-594 and 488 conjugated donkey anti-mouse and anti-rabbit (A-21203 and A-11001, Invitrogen, Carlsbad, CA). Nuclei were visualized by Hoechst nuclear stain (Invitrogen, Carlsbad, CA). Sections were mounted onto glass slides and mounted using DABCO mounting media (10 mg of DABCO (D2522; Sigma-Aldrich, St. Louis, MO) in 1 ml of PBS and 9 ml of glycerol). Slides were sealed using nail polish.
All fluorescence images were captured on Perkin Elmer ERS 6FE spinning disk confocal microscope, and images were processed and analyzed in Volocity version 6.1.1 software (Perkin Elmer, Shelton, CT).
Quantitative real-time PCR analysis
Quantitative real-time (qRT) PCR analysis of RNA isolated from embryonic day 9.5 embryos was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA,) with the CFX96 real-time PCR detection system (Bio-Rad) as previously reported
. Primer pairs (from 5′ to 3′) used for qRT-PCR analysis were as follows: Patched-1: GCCAAGCCCTAAAAAAAT and ACCACAATCAATCTCCTG (previously reported by Croyle et al.; Gli1: TCGACCTGCAAACCGTAATCC and TCCTAAAGAAGGGCTCATGGTA. The following primers for peptidylprolyl isomerase A (Ppia) were used as an internal control: CAGACGCCACTGTCGCTTT and TGTCTTTGGAACTTTGTC (both Gli and Ppia primers previously reported by Hellstrom et al.). Samples were run in triplicate using RNA from at least three different embryos per genotype.
The difference in gene expression between Cluap1
embryos was assessed using Student’s t-test on log-transformed values of the relative normalized quantity of template. Significance was established at P < 0.01. All calculations were performed using Microsoft Excel.