Telomerase reverse transcriptase (TERT) is the protein component of telomerase and combined with an RNA molecule, telomerase RNA component, forms the telomerase enzyme responsible for telomere elongation. Telomerase is essential for maintaining telomere length from replicative attrition and thus contributes to the preservation of genome integrity. Although diverse mouse models have been developed and studied to prove the physiological roles of telomerase as a telomere-elongating enzyme, recent studies have revealed non-canonical TERT activities beyond telomeres. To gain insights into the physiological impact of extra-telomeric roles, this review revisits the strategies and phenotypes of telomerase mouse models in terms of the extra-telomeric functions of telomerase.
Animals ; Mice ; Mice, Knockout ; Telomerase/genetics/*metabolism ; Telomere/metabolism

OBJECTIVE: To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice. METHODS: In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H^-/--Thy1⁺(KO-GFP) and CACNA1H^+/+-Thy1⁺ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed. RESULTS: In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F_{1, 14}=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F_{1, 14}=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05). CONCLUSION CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.
Animals ; Autistic Disorder/genetics* ; Calcium Channels, T-Type/genetics* ; Gene Knockout Techniques ; Hippocampus ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons

This study was aimed to propagate and identify the prdm1 gene-knockout mice, so as to lay the foundation for studying Blimp-1 protein. Two kinds of transgenic homozygous mice with B6.prdm1(flox/flox) and B6.Lck-Cre were feed and propagated; after successful propagating, the first passage mice were obtained; after the first passage mice were copulated once again, the genotypes were obtained as follows: B6. prdm1(wild/wild). Lck-Cre, B6. prdm1(wild/wild), B6.prdm1(flox/flox). Lck-Cre, B6.prdm1(flox/wild). Lck-Cre, B6.prdm1(flox/flox), B6. prdm1(flox/wild). The genomic DNA of second passage mice was extracted, the Cre and loxp gene fragments were amplified by PCR, then the size of Cre and loxp genomic DNA were detected by agarose gel electrophoresis. The mice with B6.prdm1(flow/flox). Lek-Cre were used as conditionally prdm1-knockout mice, B6.prdm1(flox/wild). Lck-Cre mice, B6.prdm1(flox/flox) and B6 mice were used as controls. The spleen T lymphocytes and B lymphocytes were sorted by using magnetic beads, the blimp-1 target protein was identified by Western blot. The results showed that the two transgenic homozygous mice had the ability to reproduce, and the separation ratio of second passage mice generated from propagation of their offspring cach other meet Mendelian laws, and the prdm1 gene-knockout mice also could successfully obtained. It is concluded that the application of Cre-loxp system may successfully obtain plentiful prdm1 gene-knockout mice.
Animals ; Genotype ; Mice ; Mice, Inbred C57BL ; genetics ; Mice, Knockout ; genetics ; Reproduction ; Transcription Factors ; genetics

Establishing a suitable animal model is important for studying the mechanism of inflammatory bowel disease (IBD) and exploring new therapeutic approaches. Although a large number of IBD single gene knockout animal models have been established, single knockout of certain genes associated with human IBD susceptibility does not manifest symptoms of IBD or manifest extremely milder symptoms, while composite animal models based on other modeling factors can better simulate the clinical features of IBD. This article mainly introduces three novel composite animal models and elaborates the possible pathogenesis of each composite model:animal models established by gene double knockout have more obvious and earlier symptoms than single-knockout models; single gene knockout model with Helicobacter infection can help to study the role of microbial infections in the pathogenesis of IBD; on the basis of gene knockout, specific deletion of certain immune cells can be used to study the role of the immune cells in the development of IBD. Among the above composite animal models, double knockout mice may be important animal models for IBD study.
Animals ; Disease Models, Animal ; Gene Knockout Techniques ; Humans ; Inflammatory Bowel Diseases ; genetics ; immunology ; Mice, Knockout ; Research

OBJECTIVETo breed estrogen receptor beta (ERbeta) gene knock-out female mice for studying postmenopausal osteoporotic fracture.

METHODSThree pairs of ERbeta gene knock-out mice were bred for 3 months, and 14 2-month-old female wild-type C57BL/6J mice with the same genetic background were paired at the ratio of 2:1 and mated with the male ERbeta gene knock-out homozygote mice. After further breeding to obtain sufficient number of mice, the genome DNA was extracted from the tail of the mice for genotyping by PCR. Ten 4-month-old female filial mice with ERbeta gene knock-out and 10 wild-type female mice were randomly selected and sacrificed, and the right proximal tibiae were removed and subjected to micro CT for measuring the parameters of trabecular bone histomorphometry.

RESULTSA total of 340 filial generation mice were reproduced in 2 months and genotypic identification revealed a proportion of ERbeta+ or + mice of 23.5%, ERbeta+ or - mice of 48.27 percent; and homozygous mutant (ERbeta- or -) mice of 28.3% (in which 54 were female). The MicroCT data revealed that the micro-architecture of the proximal tibiae was significantly different between ERbeta gene knock-out mice selected from the filial generation and wild type mice (P<0.05).

CONCLUSIONIt is feasible to breed ERbeta knock-out female mice by introducing female wild-type mice to pair and mate with ERbeta knock-out homozygote male mice. This approach allows breeding of sufficient number of female ERbeta knock-out mice as the animal models for studying the role of ERbeta.

Animals ; Breeding ; DNA ; analysis ; Estrogen Receptor beta ; genetics ; Female ; Gene Knockout Techniques ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout

OBJECTIVETo study the characteristics of mandible and condyle in Dmp1 gene knockout mice, and to investigate the role of Dmp1 in the osteogenesis and mineralization of bone and cartilage.

METHODSDmp1-/-mice were executed at birth, 2 weeks, 2 months, 3 months and 5 months, and the mandible was taken out for physical, radiography, transmission electron microscopic, and histological examination. The difference between Dmp1 knockout mouse (ko) and wild type mouse (wt) in bone development, bone densitometry and histology were compared.

RESULTSThere were obvious changes in the mandible and condyle of Dmp1-/-mouse, such as incomplete ossification, low density, decreased volume and condyle cartilage degeneration.

CONCLUSIONSDmp1 is the key factor in the formation of growth plates and secondary ossification center, and plays an important role in the process of bone and cartilage formation and bone nodule remodeling. Dmp1 may be the candidate gene that controls the development of mandible and cartilage.

Animals ; Bone Demineralization, Pathologic ; genetics ; pathology ; Chondrogenesis ; genetics ; Extracellular Matrix Proteins ; genetics ; Gene Knockout Techniques ; Mandible ; pathology ; Mandibular Condyle ; pathology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Osteogenesis ; genetics

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2 mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2 mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.
Animals ; CRISPR-Cas Systems ; Female ; Gene Knockout Techniques ; Gene Targeting ; Male ; Mice ; Mice, Knockout ; RNA, Guide ; genetics

OBJECTIVETo establish a mice model with selective inactivation adenosine A2A receptors (A2ARs) in peripheral white blood cells (PWBC).

METHODSA2ARs were selectively inactivated in PWBCs by transplanting bone marrow cells (BMCs) from A2AR knockout (KO) mice into their wild type (WT) littermates after a single total body irradiation of 9.5 Gy or fractionated total body irradiation of 6.2 Gy x 2. The efficiency of reconstitution of bone marrow-derived cells in chimeric mice was assessed.

RESULTSPCR band patterns changed from the recipient pattern (one band of 330 bp) to the donor (two bands of 300 and 330 bp) pattern. Immunohistochemistry analysis showed that 10.21% of cells were A2AR+ in PWBCs in KO--> WT mice, whereas 96.72% of cells were A2AR+ in WT mice. The survival rates of mice irradiated with 6.2 Gy x 2 and transplanted with more than 6 x 10(6) BMCs were about 91%.

CONCLUSIONA murine model of selective inactivation adenosine A2A receptors in PWBCs was established successfully.

Animals ; Gene Deletion ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; Receptor, Adenosine A2A ; genetics

OBJECTIVETo evaluate the effect of water channel aquaporin 4 (AQP4) on bleomycin-induced lung fibrosis in mice.

METHODSIn wild type and AQP4 gene knockout (AQP4-/-) mice, lung fibrosis was induced by injection of bleomycin (3 mg/kg) into the trachea and saline injection was used as a control. At d3, 7, 14, 28 after bleomycin-treatment, mice were randomly sacrificed in batch and the lung coefficient was determined. Serum levels of TGF-β1 and TNF-α were measured by ELISA and hydroxyproline contents in lung tissue were determined by Alkaline hydrolysis method. H-E staining and Masson's staining were performed to examine the pathological changes of lung tissues after bleomycin-treatment.

RESULTSOn d14 after bleomycin-treatment, the lung coefficients in wild type mice and AQP4-/- mice were 1.9-fold (12.69 ± 6.05 vs 6.80 ± 0.82, q=4.204, P<0.05) and 2.3-fold (14.05 ± 5.82 vs 6.05± 0.58, q=5.172, P<0.01) of that in control, respectively, but no significant difference was found between wild type and AQP4-/- mice in the lung coefficient value (P>0.05). The hydroxyproline contents in the lung increased after bleomycin-treatment; on d28, the lung hydroxyproline contents in wild type and in AQP4-/- mice were 1.55-fold (0.85 ± 0.22 g/mg vs 0.55 ± 0.14 μg/mg, q=4.313, P<0.05) and 1.4-fold (0.84 ± 0.13 μg/mg vs 0.60 ± 0.14μg/mg, q=4.595，P<0.05) of that in control, respectively, but no significant difference was noticed between wild type and AQP4-/- mice in lung hydroxyproline contents. There was a tendency that serum TGF-β1 and TNF-α levels increased in bleomycin-treated mice, but no significant difference was found between wild type and AQP4-/- mice. AQP4-knockout showed no effects on pathological changes of lung tissues with H-E staining and Masson's staining in mice with bleomycin-induced lung fibrosis.

CONCLUSIONAQP4 might not be involved in bleomycin-induced lung fibrosis in mice.

Animals ; Aquaporin 4 ; genetics ; Bleomycin ; toxicity ; Male ; Mice ; Mice, Knockout ; Pulmonary Fibrosis ; chemically induced ; genetics

A large number of putative risk genes for autism spectrum disorder (ASD) have been reported. The functions of most of these susceptibility genes in developing brains remain unknown, and causal relationships between their variation and autism traits have not been established. The aim of this study was to predict putative risk genes at the whole-genome level based on the analysis of gene co-expression with a group of high-confidence ASD risk genes (hcASDs). The results showed that three gene features - gene size, mRNA abundance, and guanine-cytosine content - affect the genome-wide co-expression profiles of hcASDs. To circumvent the interference of these features in gene co-expression analysis, we developed a method to determine whether a gene is significantly co-expressed with hcASDs by statistically comparing the co-expression profile of this gene with hcASDs to that of this gene with permuted gene sets of feature-matched genes. This method is referred to as "matched-gene co-expression analysis" (MGCA). With MGCA, we demonstrated the convergence in developmental expression profiles of hcASDs and improved the efficacy of risk gene prediction. The results of analysis of two recently-reported ASD candidate genes, CDH11 and CDH9, suggested the involvement of CDH11, but not CDH9, in ASD. Consistent with this prediction, behavioral studies showed that Cdh11-null mice, but not Cdh9-null mice, have multiple autism-like behavioral alterations. This study highlights the power of MGCA in revealing ASD-associated genes and the potential role of CDH11 in ASD.
Animals ; Autism Spectrum Disorder/genetics* ; Brain ; Cadherins/genetics* ; Gene Expression ; Mice ; Mice, Knockout