1.Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure.
Shijuan GAO ; Guangming FANG ; Yanhong ZHANG ; Jie DU
Chinese Journal of Biotechnology 2023;39(4):1644-1654
To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.
Animals
;
Mice
;
Mice, Knockout
;
CRISPR-Cas Systems
;
Blood Pressure
;
Gene Knockout Techniques
;
Zygote
2.Mildly Increased Mechanical Nociceptive Sensitivity in REV-ERBα Knock-out Mice.
Jaehyun LEE ; Hyoung Gon KO ; Kyungjin KIM ; Bong Kiun KAANG
Experimental Neurobiology 2016;25(6):342-346
Nociception is one of the most complex senses that is affected not only by external stimulation but also internal conditions. Previous studies have suggested that circadian rhythm is important in modulating nociception. REV-ERBα knock-out (KO) mice have disrupted circadian rhythm and altered mood-related phenotypes. In this study, we examined the role of REV-ERBα in inflammatory nociception. We found that the nociceptive sensitivity of KO mice was partially enhanced in mechanical nociception. However, this partial alteration was independent of the circadian rhythm. Taken together, deletion of REV-ERBα induced a mild change in mechanical nociceptive sensitivity but this alteration was not dependent on the circadian rhythm.
Animals
;
Circadian Rhythm
;
Mice
;
Mice, Knockout*
;
Nociception
;
Phenotype
3.The Development of NKT Cells in Thymus is Defective in CD45 Knockout Mice.
Doo Hyun JUNG ; David H MARGULIES
Korean Journal of Immunology 2000;22(3):117-121
No abstract available.
Animals
;
Mice
;
Mice, Knockout*
;
Natural Killer T-Cells*
;
Thymus Gland*
4.Extracting Extra-Telomeric Phenotypes from Telomerase Mouse Models.
Young Hoon SUNG ; Muhammad ALI ; Han Woong LEE
Yonsei Medical Journal 2014;55(1):1-8
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
5.14-3-3γ Haploinsufficient Mice Display Hyperactive and Stress-sensitive Behaviors
Do Eon KIM ; Chang Hoon CHO ; Kyoung Mi SIM ; Osung KWON ; Eun Mi HWANG ; Hyung Wook KIM ; Jae Yong PARK
Experimental Neurobiology 2019;28(1):43-53
14-3-3γ plays diverse roles in different aspects of cellular processes. Especially in the brain where 14-3-3γ is enriched, it has been reported to be involved in neurological and psychiatric diseases (e.g. Williams-Beuren syndrome and Creutzfeldt-Jakob disease). However, behavioral abnormalities related to 14-3-3γ deficiency are largely unknown. Here, by using 14-3-3γ deficient mice, we found that homozygous knockout mice were prenatally lethal, and heterozygous mice showed developmental delay relative to wild-type littermate mice. In addition, in behavioral analyses, we found that 14-3-3γ heterozygote mice display hyperactive and depressive-like behavior along with more sensitive responses to acute stress than littermate control mice. These results suggest that 14-3-3γ levels may be involved in the developmental manifestation of related neuropsychiatric diseases. In addition, 14-3-3γ heterozygote mice may be a potential model to study the molecular pathophysiology of neuropsychiatric symptoms.
Animals
;
Anxiety
;
Brain
;
Heterozygote
;
Mice
;
Mice, Knockout
;
Williams Syndrome
6.Breeding and identification of estrogen receptor beta gene knock-out mice.
Xiao-hua PAN ; Yu-gang WANG ; Ge ZHANG ; Zhong LIU ; Wan-cheng ZENG ; Jia-Kai CHEN ; Yong DAI ; Ling QIN
Journal of Southern Medical University 2010;30(1):153-156
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
7.Effect of arachidonic acid cytochrome P450ω hydroxylase Cyp4a14 gene knockout on skeletal muscle regeneration after injury.
Cong-Cong ZHANG ; Ying-Chun HAN ; Nai-Xuan CHENG ; Jie DU
Acta Physiologica Sinica 2021;73(4):577-583
The objective of this study was to explore the roles of arachidonic acid cytochrome P450ω hydroxylase CYP4A14 in skeletal muscle regeneration after injury. Wild-type (WT) control mice and Cyp4a14 knockout (A14
Animals
;
Arachidonic Acid
;
Cytochromes
;
Gene Knockout Techniques
;
Mice
;
Mice, Inbred C57BL
;
Mice, Knockout
;
Mixed Function Oxygenases
;
Muscle, Skeletal
;
Regeneration
8.Effects of CACNA1H gene knockout on autistic-like behaviors and the morphology of hippocampal neurons in mice.
Cui JIAO ; Jian Mei WANG ; Hai Xia KUANG ; Zhi Hong WU ; Tao LIU
Journal of Peking University(Health Sciences) 2022;54(2):209-216
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 (F1, 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 (F1, 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
9.Research progress on composite animal models of inflammatory bowel disease based on gene knockout.
Journal of Zhejiang University. Medical sciences 2018;47(6):665-670
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
10.Progress in gene knockout mice.
Chinese Journal of Biotechnology 2019;35(5):784-794
The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.
Animals
;
Disease Models, Animal
;
Embryonic Stem Cells
;
Gene Knockout Techniques
;
trends
;
Gene Targeting
;
trends
;
Homologous Recombination
;
Humans
;
Mice
;
Mice, Knockout