Investigators used a commercially supplied C57BL/6N CRISPR-Cas9 model in which a 1610 bp fragment containing Ttc22 exons 2 and 3 was deleted. PCR sequencing and RNA-seq confirmed removal of the targeted exon region. The deleted segment encodes the TPR4 motif of the mouse protein, which corresponds to human TPR3 by sequence alignment provided in the source. A rabbit polyclonal antibody against human TTC22, previously developed by the group, bound both full-length and TPR-deleted Flag-tagged mouse constructs expressed in HEK293T cells. Western blot analysis of colon tissue showed robust Ttc22 protein in wild-type and heterozygous mice, but no detectable signal in homozygous knockout (Ttc22 -/-) colon samples, indicating effective loss of protein expression in the targeted tissue.
The genotyping approach pooled three primers to amplify both wild-type and deleted alleles in a single assay. Expected amplicon patterns differed by genotype, and sequencing confirmed the engineered deletion. The authors reported that the exon 2&3 deletion eliminated expression of the exon region shared by both annotated Ttc22 transcript variants, as visible in IGV plots from RNA-seq reads of colon samples.
The study team tracked body weight, general health, and reproductive outcomes across Ttc22 +/+, Ttc22 +/-, and Ttc22 -/- mice. Body weight was measured weekly from 4 to 42 weeks of age, and animals were inspected twice daily for activity and welfare. The cohort showed no genotype-dependent differences in body weight trajectories or overt developmental abnormalities. Mating tests were performed using matched genotype pairs and recorded pup numbers at birth; these fertility assessments produced no genotype-related deficits. Spontaneous tumor incidence during the observation window was not increased in Ttc22-deficient mice. The investigators therefore reported that Ttc22 inactivation did not affect growth, development, fertility, or spontaneous tumor formation under the described husbandry and observation conditions.
The source detailed a separate high-fat diet challenge in female mice, where a 45% fat-calorie diet was provided from 4 weeks of age for 42 weeks. Animals had ad libitum access to food and water and were housed under controlled conditions. The article presented this as part of phenotyping but did not report genotype-specific metabolic endpoints or differential weight gain attributable to Ttc22 status in the source text beyond stating no overt phenotype; specific diet-response data and statistical comparisons were not enumerated in the provided extract.
To probe susceptibility to chemically induced colon tumorigenesis, male mice of each genotype received a single intraperitoneal dose of azoxymethane (AOM) followed by cycles of dextran sodium sulfate (DSS) in drinking water. The combined AOM/DSS protocol was repeated three times and animals were sacrificed in the 24th–27th experimental weeks for colon examination and pathologic assessment. The investigators reported no differences in the incidence of AOM/DSS-induced COAD among Ttc22 +/+, Ttc22 +/-, and Ttc22 -/- mice. Gross colon length measurements, mucosal inspection, and histopathology were performed; pathology review was conducted by senior pathologists. Two mice in the entire experimental set died early during AOM/DSS experiments for undetermined causes, but the remaining animals completed the protocol and were analyzed. The source did not present detailed tumor counts, sizes, or effect sizes in the text excerpt provided.
RNA sequencing of colon tissue from representative male mice of each genotype revealed differential gene expression patterns in Ttc22 -/- animals. The investigators observed upregulation of gene sets associated with protein folding and cellular stress responses. The authors interpreted these changes as an adaptive host response that could compensate for the loss of Ttc22 function. The study deposited raw RNA-seq data at GEO under accession GSE275363. The article also referenced prior work from the group showing that human TTC22v1 can modulate m6A-mediated expression of WTAP and SNAI1 via RPL4 interaction and promote metastatic phenotypes in human colon cancer models; however, analogous phenotypes were not replicated as genotype-dependent tumor susceptibility in this mouse model under the tested conditions.
The authors proposed that genetic redundancy or robustness likely explains the lack of observable phenotypes after Ttc22 deletion in the C57BL/6N background. The source did not report specific effect sizes or p values for the AOM/DSS tumor endpoints within the provided text, nor did it detail whether alternate environmental stresses or different genetic backgrounds were tested. The increased expression of stress- and folding-related genes in knockout colons suggests compensation, but the mechanistic pathways and whether compensation is cell-type specific were not resolved in the presented material. Raw RNA-seq data are available for reanalysis (GSE275363), which may permit further interrogation. The study did not report long-term lifespan beyond the defined observation windows, nor did it test other tumor models or conditional tissue-specific deletions that might reveal context-dependent roles for Ttc22.