---
title: "Ttc22 knockout mice show no overt developmental or colon cancer phenotypes"
id: "plos-one-7-no-phenotypes-in-ttc22-knockout-mice"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-7-no-phenotypes-in-ttc22-knockout-mice"
content_type: "clinical_feed_article"
specialty: "Oncology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0361340"
published_at: "2026-10-23T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Ttc22 knockout mice show no overt developmental or colon cancer phenotypes
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-7-no-phenotypes-in-ttc22-knockout-mice
- **Specialty:** [Oncology](https://medichelpline.com/clinical-feed/oncology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0361340)
- **Published At:** 2026-10-23T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The study used a commercially available C57BL/6N CRISPR-Cas9 mouse model in which **Ttc22** exons 2 and 3 (encoding the TPR4 domain) were deleted. - RNA sequencing and PCR confirmed the 1610 bp deletion spanning exons 2&3 in knockout alleles and loss of the exon region in homozygous mice. - A rabbit polyclonal antibody raised against TTC22 bound full-length and TPR-deleted mouse Ttc22 constructs in HEK293T cells; colon tissue from Ttc22 -/- mice lacked detectable Ttc22 protein by Western blot. - Across lifespan monitoring (4–42 weeks) the investigators observed no differences in body weight, growth, fertility, or spontaneous tumor incidence between Ttc22 +/+, Ttc22 +/-, and Ttc22 -/- mice. - Under an AOM/DSS chemical carcinogenesis protocol there were no differences in colon adenocarcinoma (COAD) incidence between genotypes. - Transcriptomic profiling of colon tissue identified upregulation of genes involved in protein folding and stress responses in Ttc22 -/- mice, suggesting activation of adaptive compensatory pathways. - The authors conclude **Ttc22** inactivation in C57BL/6N mice is phenotypically silent under the tested conditions, consistent with genetic redundancy or robustness. - RNA-seq raw data are available at GEO accession GSE275363. - The source did not report detailed effect sizes for AOM/DSS tumor outcomes, nor did it provide long-term follow-up beyond the stated observation windows for all experiments.
## Clinical Analysis & Structured Key Points
No phenotypes in Ttc22 knockout mice | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract The TTC22 gene encodes a protein containing seven tetratricopeptide repeats (TPRs), which mediate protein‒protein interactions as chaperones. We previously reported that the level of TTC22 transcript variant 1 ( TTC22v1 ) was downregulated in human colon adenocarcinoma (COAD) and that TTC22 upregulated m6A-mediated WTAP and SNAI1 expression via the TTC22‒RPL4 interaction and subsequently promoted COAD metastasis. Thus, a commercially available C57BL/6N mouse model in which the Ttc22 exon 2&3 encoding the TPR4 (equal to the human TTC22 TPR3) domain was knocked out via CRISPR-Cas9 was used to evaluate the contribution of Ttc22 to the development of mice and COAD. To determine the status of Ttc22 expression in Ttc22 knockout mice, we prepared a rabbit polyclonal antibody against the mouse Ttc22 protein, which could bind both wild-type Ttc22 and TPR4-deleted Ttc22 proteins overexpressed in HEK293T cells, as determined by Western blot analysis. Ttc22 proteins were detected in colon tissue samples from wild-type mice and Ttc22 hemi-knockout ( Ttc22 +/- ) mice but not in those from fully Ttc22 knockout ( Ttc22 -/- ) mice. Unfortunately, the long-term observation results demonstrated that Ttc22 knockout (KO, including Ttc22 +/- or Ttc22 -/- ) did not affect the body weight, development, fertility, or spontaneous tumor incidence of male or female mice. No differences in the incidence of AOM/DSS-induced COAD were observed between these mouse groups, although Ttc22 deletion partially resulted in the upregulation of genes related to the host adaptive response to inflammation in the colon mucosa. In conclusion, no phenotypes were induced by Ttc22 inactivation in C57BL/6N mice. Citation: Liu J, Gu L, You A, Yuan H, Zhou J, Tian W, et al. (2026) No phenotypes in Ttc22 knockout mice. PLoS One 21(10): e0361340. https://doi.org/10.1371/journal.pone.0361340 Editor: Zhanzhan Li, Xiangya Hospital Central South University, CHINA Received: April 1, 2026; Accepted: October 7, 2026; Published: October 23, 2026 Copyright: © 2026 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All mice RNA-sequencing raw data for this study are available from the Gene Expression Omnibus database with accession number GSE275363 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE275363) . Funding: This work was supported by grants from the Beijing Hospital Authority Mission Plan (SML20191101) to DD and from the Science Foundation of Peking University Cancer Hospital (grant #2022-8) to TW. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: COAD, Colon adenocarcinoma; Ttc22, Tetratricopeptide repeat domain 22; AOM, Azoxymethane; DSS, Dextran sodium sulfate; m6A, N6-methyladenosine; LDL, Low-density lipoprotein; TCGA, The Cancer Genome Atlas; Ttc22-KO, Ttc22 knockout; SPF, Specific pathogen-free; FPKM, Fragments per kilobase of transcript per million mapped reads; FC, Fold Change; GO, Gene Ontology; LC‒MS/MS, Liquid chromatography–tandem mass spectrometry; NC, Negative control; PC, Positive control; DTT, Dithiothreitol; SDS-PAGE, Sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PBS, Phosphate-buffered saline; SD, Standard deviation Introduction The human TTC22 protein contains seven tetratricopeptide repeats (TPRs), which mediate protein‒protein interactions and are involved in multiple biological processes, including the movements of cilia and flagella and the migration of human cells [ 1 – 5 ]. A pilot study revealed that the TTC22 gene was among the top ten downregulated genes in the fetuses of obese women compared with lean women in the second trimester and that low TTC22 expression in mothers may be related to fetal neurodevelopment and metabolism [ 6 ]. Differentially methylated regions of the TTC22 gene are associated with hand grip strength [ 7 ]. DNA sequencing and transcriptome data analyses in 3,203 subjects suggested that low-frequency variants of TTC22 were associated with low-density lipoprotein (LDL) cholesterol [ 8 ]. These phenomena imply that TTC22 may be involved in physiological metabolism. However, the causality between TTC22 alterations and metabolism is not known. According to data from the Human Protein Atlas and The Cancer Genome Atlas (TCGA), the TTC22 gene is comprehensively expressed in epithelial cells in the normal gut mucosa at the middle level and is extensively downregulated in gastrointestinal cancer tissues [ 9 , 10 ]. We initially reported that TTC22 variant 1 ( TTC22v1 ) mRNA is targeted by miR663a , an anti-inflammatory miRNA in the colon [ 11 ]. TTC22v1 mRNA expression is significantly lower in human colon adenocarcinoma (COAD) than in paired normal tissue samples, and high TTC22 expression increases the risk of colon cancer metastasis. Our further study revealed that TTC22 increased m6A-mediated WTAP and SNAI1 expression via TTC22-RPL4 binding and subsequently promoted COAD metastasis in vitro and in vivo [ 12 ], suggesting a potential role for TTC22 in the development and progression of COAD. Therefore, we further studied the effects of Ttc22 on the development of mice and their susceptibility to the chemical carcinogen AOM/DSS via a genetically established and commercially available C57BL gene knockout mouse model in which Ttc22 exons 2 and 3, which encode the TPR4 domain of the mouse Ttc22 protein, were knocked out via CRISPR‒Cas9. While qRT-PCR and Western blot analyses confirmed the complete absence of Ttc22 mRNA and protein in the colon tissues of mice with a homozygous deletion of two Ttc22 alleles ( Ttc22 -/- ), unexpectedly, no differences in the development, growth, fertility, or AOM/DSS susceptibility of the mice were induced by Ttc22 knockout. Transcriptomic analysis of colon tissues revealed significant upregulation of genes involved in protein folding and stress response in Ttc22 -/- mice, indicating that loss of Ttc22 induces a broad adaptive response that may compensate for its absence. Our findings demonstrate that Ttc22 is dispensable under physiological conditions because of gene redundancy or genetic robustness, highlighting the importance of compensatory mechanisms in gene function studies. Materials and methods Ttc22 exon 2&3 knockout mouse model Genetically established Ttc22 knockout (Ttc22-KO) C57BL/6N mice (strain S-KO-06335) were purchased from Cyagen (Cyagen Bio. Co., Guangzhou, China). Ttc22 knockout mice were obtained via high-throughput electroporation of fertilized eggs. The CRISPR-Cas9 system and two gRNAs (gRNA#1: 5’-ggaaagugggcauaacgccuggg-3’ and gRNA#2: 5’-uuccagagagauagcgaccgagg-3’) targeted to Ttc22 introns 1&3 were used to delete a 1610 bp fragment including Ttc22 exons 2&3 encoding the Ttc22 TPR4 domain ( Fig 1A ). After sexual maturity, the sperm were collected for cryopreservation. Through hybridization with female wild-type C57BL/6N mice, first-generation Ttc22 +/- mice were obtained and used to generate male and female mice with different Ttc22 genotypes (including Ttc22 +/+ , Ttc22 +/- , and Ttc22 -/- ), as determined by PCR sequencing via the tail DNA template and two forward primers (OuterF: 5’-tcacaacctactcaagatccagc-3’ and InnerF: 5’-caactgtagggtatagcagtccc-3’) and one common reverse primer (OuterR: 5’-ttattgcctccttggtgagttg-3’). A mouse tissue direct PCR kit (YEASEN, Shanghai, China) was used to amplify Ttc22. Both male and female Ttc22 +/+ and Ttc22 -/- mice were maintained as seed mice in an animal facility under SPF conditions at the Peking University Cancer Hospital & Institute under a 12-hr light/dark cycle at 22 ± 2°C and 50 ± 10% humidity, with ad Librium access to sterilized chow and water. Cages were supplemented with nesting materials for environmental enrichment to minimize husbandry-related stress. The seed mice were hybridized to generate experimental mouse stocks with different Ttc22 genotypes for model establishment and subsequent studies. Animal health, food intake, and spontaneous activity were monitored twice daily (morning and afternoon), and body weight was measured once a week from 4 to 42 weeks of age as a primary welfare indicator. No invasive surgical procedures or intentionally painful interventions were performed in this study; therefore, no general anesthesia or systemic analgesic agents were administered throughout the observation period. Predefined humane endpoint criteria were strictly implemented as an alternative measure to prevent and minimize potential animal suffering. Mice exhibiting severe lethargy, sustained anorexia, or >20% body weight loss prior to the scheduled endpoints were humanely euthanized via cervical dislocation, which was performed by experienced personnel promptly upon completion of the monitoring session on the same day, in accordance with prespecified humane endpoints and guidelines for the euthanasia of the animals. All procedures were performed by personnel who had undergone systemic training and who possessed extensive experience. Total 340 mice were used in this study. Two of them treated with AOM/DSS experienced early body weight loss and subsequent fatalities at the 1st and 6th experimental weeks because of undetermined causes, and the remaining 338 mice were euthanized via cervical dislocation. The animal experiments were approved by the Biomedical Ethical Committee of Peking University Cancer Hospital & Institute (Protocol No. EAEC2019−09). Download: PNG larger image TIFF original image Fig 1. Characterization of the genotypes of the Ttc22 gene in C57BL/6N mice via CRISPR-Cas9. (A) Locations of the deleted fragment (1610 bp), amplicons, gRNAs, and primers matched to the Ttc22 gene. The conservation status of Ttc22 in placental mammals, including rats, rabbits, and humans, is displayed below. (B) Characterization of Ttc22 genotypes, as determined by PCR and sequencing. The wild-type Ttc22 fragment is too long (2438 bp) to be amplified via regular PCR via the OuterF and OuterR primer sets. (C) IGV view of Ttc22 transcripts in RNA samples from the colons of three male mice with different Ttc22 genotypes, namely, Ttc22 wild-type ( Ttc22 +/+ ), heterozygously deleted ( Ttc22 +/- ), and homozygously deleted ( Ttc22 -/- ) strains, as determined by RNA sequencing. The exon 2&3 region, which is shared by both Ttc22 variants 1 and 2 and is knocked out by CRISPR-Cas9, is highlighted by a red rectangle. https://doi.org/10.1371/journal.pone.0361340.g001 High-fat diet experiment Four-week-old female C57BL/6N mice were consistently fed a high-fat diet, in which 45% of the calories were derived from fat (4.73 kcal/g H10045, Huafukang Bio. Co., Beijing, China). Additionally, a control group of four-week-old female mice was fed a regular diet. The mice were housed in a temperature-controlled room with a 12-h light/dark cycle for 42 weeks. All the mice had free access to food and water [ 13 ]. Fertility test Mating tests were carried out on 6–8-week-old mice. One male Ttc22 +/+ mouse was mated with 1 Ttc22 +/+ female mouse as the control group, and 1 male Ttc22 -/- mouse was mated with 1 Ttc22 -/- female mouse as the Ttc22-KO group for the first pregnancy. At least three mates were included in each group. The date and number of pups were recorded at birth in each cage. Induction of COAD by AOM/DSS Five male mice aged 6–8 weeks were included in each group within the same cage. The mice in the three groups, Ttc22 +/+ , Ttc22 +/- , and Ttc22 -/- were intraperitoneally administered 10 mg/kg b.w. azoxymethane (AOM) solution (1 mg/mL, freshly prepared with saline; Cat. #A5486 Sigma‒Aldrich; Merck KGaA, Darmstadt, Germany) on the 1st experimental day. Beginning on the 2nd experimental day, the mice were administered dextran sodium sulfate (DSS; Cat. #24867; Sigma) in the drinking water containing 2.5% DSS (2.5 g/100 mL) for 7 continuous days. The mice were subsequently allowed to recover for three weeks (drinking water not containing DSS). The combined AOM/DSS treatment cycle was repeated two more times [ 14 ]. From the 24th to 27th experimental weeks, the mice were sacrificed, the colon was collected, and the length of the colon was measured. The intestine was opened, and the colon mucosa was carefully examined and photodocumented. Evidence of gross pathologic changes was recorded (e.g., ulceration). Afterward, the colon tissues were fixed with 10% formalin, rolled Swiss, embedded in paraffin, and sectioned for pathological analysis. RNA or protein was extracted from a portion of fresh colon tissue with TRIzol or cell lysis buffer. The remaining tissue was stored at −80°C for subsequent studies. The roll was further fixed in 10% buffered formalin overnight at room temperature and used to prepare tissue sections (4 μm). Pathology was performed by senior pathologists. RNA sequencing Total RNA was extracted from the colon tissues of three male mice with different Ttc22 genotypes via an RNA extraction kit. RNA sequencing was performed by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA was removed from the rRNAs with an NEBNext rRNA depletion kit (Biolabs, Inc., Massachusetts, USA), which was subsequently used to construct RNA libraries via the NEBNext Ultra II directional RNA library prep kit (Biolabs). Library sequencing was performed on an Illumina NovaSeq 6000 instrument with 150 bp paired-end reads. Paired-end reads were subsequently harvested. The high-quality clean reads were aligned to the reference genome with HISAT2 software (v2.0.4), guided by the Ensembl gtf gene annotation file, and the gene level FPKM was obtained as the expression profile of the mRNAs. Fold changes and p values were calculated on the basis of the FPKM values, and the differentially expressed mRNAs were identified on the basis of |log(FC)|>=1.0 and a p value<= 0.05. GO and pathway enrichment analyses were performed on the differentially expressed mRNAs. The raw data were deposited under the Gene Expression Omnibus accession number GSE275363. m6A dot blot analysis m6A dot blot analysis was performed as we previously reported [ 12 ]. Quantification of m6A abundance via m6A-specific ELISA The total RNA m6A level was quantified with an EpiQuik m6A RNA Methylation Quantification Kit (P-9005; Epigentek Group, NY, USA) according to the manufacturer’s instructions [ 15 , 16 ]. LC‒MS/MS measurement of the m6A/rA ratio Liquid-MS/MS quantification of m6A was performed by Cloudseq Biotech, Inc. (Shanghai, China), as described previously [ 12 ]. Full-length Ttc22, ΔTPR1, and ΔTPR4 expression vectors The pCMV6-Flag full-length Ttc22 expression vector was purchased from the MiaoLing Plasmid Platform (Wuhan, China) [ 12 ]. The ΔTPR4 vector not containing 568–738 bp and the ΔTPR1 vector not containing 4–297 bp were derived from the full-length Ttc22 vector and confirmed by Sanger sequencing ( S1 – S3 Figs ). These vectors were used to transiently transfect human noncancer HEK293T cells (ATCC; Manassas, VA, USA). Western blot analysis Cells were harvested 48 hrs after transfection and in 1× cell lysis buffer [50 mmol/L Tris-HCl (pH 6.8), 100 mmol/L DTT, and 2% SDS. 0.1% bromophenol blue and 10% glycerol] was used to extract total cellular proteins. Protein electrophoresis was performed using 10% SDS-PAGE gels. The membranes were incubated with the following primary antibodies at 4°C overnight: a mouse anti-DYKDDDDK Flag monoclonal antibody (1:3000; cat. no. 66008–4-Ig, Proteintech Group, Inc. Wuhan, China), a mouse anti-Gapdh monoclonal antibody (1:10000; cat. no. 60004–1-Ig; Proteintech Group, Inc., Wuhan, China), and a rabbit anti-His-TTC22 (1–569 aa) polyclonal antibody previously raised and characterized in our own laboratory [ 12 ]. The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5000; cat.no. A0208 or A0216, Beyotime Technology, Shanghai, China) at room temperature for 50 min. Mouse colon tissue was collected and homogenized in a 1.5 mL microcentrifuge tube. Afterward, 1 mL of lysis buffer was added per 50 mg of tissue. The remaining steps were carried out as described above for the cell samples. Statistical analysis Statistical analysis was carried out via SPSS 22. 0 software (SPSS Inc., Chicago IL, USA). The data are presented as the means ± SDs of at least three independent experiments or as the medians (25th–75th percentiles). Statistical analysis methods included Student’s t test, one-way analysis of variance (ANOVA), two-way analyses of ANOVA and RM-ANOVA. The exact statistical methods are described in detail for each experiment in the Results section. All tests were two-sided. A p value <0.05 was considered to indicate statistical significance. Results Characterization of Ttc22 genotypes To evaluate the effects of the Ttc22 gene on the development of mice and COAD, a commercially available heterozygous Ttc22-KO C57BL/6N mouse model was used to generate mice with various Ttc22 genotypes, including homozygous and heterozygous Ttc22-KO ( Ttc22 -/- and Ttc22 +/- ) and its wild-type control ( Ttc22 +/+ ) mice, as described in the Methods section. The sequencing results indicated that a 1610 bp fragment, including Ttc22 exons 2 and 3, which encode the Ttc22 TPR4 domain (57 aa), was deleted via CRISPR-Cas9 and two gRNAs ( Fig 1A and 1B ). RNA sequencing confirmed the sequencing results ( Fig 1C ). To determine the expression level of the Ttc22 protein in the colons, we performed Western blot analysis using a rabbit polyclonal antibody against human TTC22 previously created at our laboratory [ 12 ]. We initially investigated whether the TTC22 antibody might also bind mouse Ttc22 and found that the TTC22 antibody efficiently bound both wild-type Flag-Ttc22 and its ΔTPR1 and ΔTPR4 variants ( Figs 2A and S1 – S3 ) overexpressed in HEK293T cells by Western blotting ( Fig 2B ). These results were consistent with those obtained with the anti-Flag antibody. However, Ttc22 was detected only in the colon tissues from Ttc22 +/+ and Ttc22 +/- male mice, and no Ttc22 was detected in the colon tissues from Ttc22 -/- mice ( Fig 2C ), indicating complete loss of Ttc22 expression in the colon. We further checked the knockout status of the Ttc22 alleles in the colon tissues from these mice. To simultaneously amplify both wild-type Ttc22 and its exon 2&3-deleted variant by the same PCR, we pooled three primers (OuterF, InnerF, and OuterR) together. As expected, the 828 bp PCR products were amplified only by OuterF and OuterR primers from the genetic DNA of Ttc22 -/- and Ttc22 +/- mice and were not amplified from the genetic DNA of Ttc22 +/+ mice (1610 bp + 828 bp = 2438 bp; too long to be amplified). In contrast, the 568 bp PCR products were amplified only by the InnerF and OuterR primers in Ttc22 +/+ and Ttc22 +/- male mice and not in Ttc22 -/- mice ( Fig 1D ). Collectively, these results indicate that only Ttc22 exons 2 and 3 are knocked out in these mice, which may lead to complete loss of Ttc22 expression. Download: PNG larger image TIFF original image Fig 2. Characterization of the knockout status of the Ttc22 gene in the mouse colon. (A) Illustration of wild-type Ttc22 and its TPR1- or TPR4-deleted (ΔTPR1 or ΔTPR4) expression vectors. (B) Western blot images of the full-length (FL)/wild-type Ttc22 protein and its ΔTPR1 or ΔTPR4 variant overexpressed in HEK293T cells.
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