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Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, timeconsuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains. [ABSTRACT FROM AUTHOR]

AbstractAim:To evaluate whether insulin intervention could affect the metabolic and endocrine functions of adipose tissue.Methods:C57BL/6 mice were fed on a high-fat-diet for 12−16 weeks to induce insulin resistance. Insulin intervention was administered in the high-fat-diet mice for 4 weeks at 12 weeks (early insulin treatment) or 16 weeks (late insulin treatment). Intraperitoneal glucose tolerance tests were performed before and after insulin treatment. Expression levels of factors involved in the triglyceride synthesis and endocrine functions of adipose tissue including phosphoenolpyruvate carboxykinase (PEPCK-C), fatty acid synthase (FAS), aquaporin 7 (AQP7), adiponectin, visfatin, and interleukin-6 (IL-6) were determined by Western blot.Results:In the obese mice, glucose tolerance was impaired; triglyceride content was increased in the liver tissue; protein expression of FAS and adiponectin was decreased; expression of visfatin was increased in adipose tissue. After 4-week insulin treatment, glucose tolerance was improved; triglyceride content was decreased in the liver and skeletal muscle; expression of PEPCK-C, FAS, and adiponectin was increased in the adipose tissue; IL-6 and AQP7 expression was reduced in the fat. Early insulin treatment had better effect in increasing the expression of FAS and PEPCK-C and decreasing the expression of IL-6.Conclusion:These results indicate that insulin can target adipocytes for improvement of insulin sensitivity through stimulating triglyceride synthesis and partly improving endocrine functions. [ABSTRACT FROM AUTHOR]

We investigated the effects of apocynin on high-fat diet- (HFD-) induced insulin resistance in C57BL/6 mice. After 12 weeks of HFD, the mice that exhibited insulin resistance then received 5 weeks of apocynin (2.4 g/L, in water). Following apocynin treatment, fasting glucose, insulin, and glucose tolerance test showed significant improvement in insulin sensitivity in HFD-fed mice. We demonstrated that serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and leptin were remarkably reduced with apocynin treatment. We also found that mRNA expression of TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1) in the liver and mRNA expression of TNF-α, IL-6, MCP-1, and leptin in adipose tissue were suppressed by apocynin. Furthermore, the activity of transcription factor NF-κB in the liver was significantly suppressed with apocynin treatment. These results suggest that apocynin may reduce inflammatory factors in the blood, liver, and adipose tissue, resulting in amelioration of insulin resistance in HFD-fed mice. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the effects of chimerism on the mice heart transplanted survival with the bone marrow infusion. Methods: Bone marrow cells (BMCs) were obtained from BALB/c mice. These BMCs were injected into the irradiated (2Gy-Co60) C57BL/6 mice through femoral vein. Then Group a mice were treated with Cyclosporine (1 mg/kg) for 21 days and Group B were not treated with Cyclosporine. Group C were treated as the control group without BMCs infusion. Group D were treated with Cyclosporine (1 mg/kg) for 21 days pre heart transplantation without BMCs infusion. After 21 days, the C57BL/6 mice received heart allografts from BALB/c. To determine the degree of chimerism in BMCs infusion recipients, peripheral blood were isolated on day 7, 14, 21. Allografts were harvested 10 days after heart transplantation for the histological 'analysis. Results: (1) Chimerism detected in the peripheral blood of Group A mice on day 7 after BMCs infusion was 6.1 ± 2.5%, on day 14 was 15.4 ± 2.9% and on day 21 was 10.7 ± 2.6%. For the Group B mice on day 7 after BMCs infusion, the chimerism was 2.8 ± 1.1%, on day 14 was 11.2 ± 4.8% and on day 21 was 7.4 ± 3.7%. For the Groups C and D mice, no chimerism was observed. Group A mice had the tendency toward improved level of chimerism than Group B mice. (2) The survival time of Group A (n = 6) was 13.0 ± 1.4 days which was significantly longer than Group B (n =6) with the survival time was 8.5 ± 1.3 days (p<0.001), also longer than the mice in Groups C and D, the survival time of which were 10.0 ± 1.3 days (p = 0.008) and 9.44 ± 1.1 days (p =0.004). There is no significant difference among Groups B, C, and D. (3) The HE staining showed the much more seriously heart rejection in Groups B, C and D than Group A. Conclusions: The chimerism was found in the BMCs infusion groups. Without the CsA treatment combined with chimerism could not protect the transplanted heart. There was no obvious evidence showed that the chimerism alone could improve the survival time of cardiac allografts in mice. [ABSTRACT FROM AUTHOR]