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Establishment of liver specific glucokinase gene knockout mice: a new animal model for screening anti-diabetic drugs

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Acta Pharmacologica Sinica 2004年第12期25卷 93-99页

作者： Ya-li ZHANG, Xiao-hong TAN2, Mei-fang XIAO, Hui LI, Yi-qing Mao, Xiao YANG2, Huan-ran TAN3 Department of Pharmacology, School of Basic Medical Science, Peking University Health Science Center, Beijing 100083 2Institute of Biotechnology Academy ofMilitary Medical Science, Beijing 100071, China Department of Pharmacology School of Basic Medical Science Peking University Health Science Center 北京 100083 Institute of Biotechnology Academy of Military Medical Science 北京 100071

AIM: To characterize the liver-specific role of glucokinase in maintaining glucose homeostasis and to create an animal model for diabetes. METHODS: We performed hepatocyte-specific gene knockout of glucokinase in mice using Cre-loxP gene targeting strategy. First, two directly repeated loxP sequences were inserted to flank the exon 9 and exon 10 of glucokinase in genomic DNA. To achieve this, linearized targeting vector was electroporated into ES cells. Then G418- and Gancyclovir-double-resistant clones were picked and screened by PCR analysis and the positives identified by PCR were confirmed by Southern blot. A targeted clone was selected for microinjection into C57BL/6J blastocysts and implanted into pseudopregnant FVB recipient. Chimeric mice and their offspring were analyzed by Southern blot. Then by intercrossing the Alb-Cre transgenic mice with mice containing a conditional gk allele, we obtained mice with liver-specific glucokinase gene knockout. RESULTS: Among 161 double resistant clones 4 were positive to PCR and Southern blot and only one was used for further experiments. Eventually we generated the liver specific glucokinase knockout mice. These mice showed increased glucose level with age and at the age of 6 weeks fasting blood glucose level was significantly higher than control and they also displayed impaired glucose tolerance. CONCLUSION: Our studies indicate that hepatic glucokinase plays an important role in glucose homeostasis and its deficiencies contribute to the development of diabetes. The liver glucokinase knock- out mouse is an ideal animal model for MODY2, and it also can be applied for screening anti-diabetic drugs.

关键词： animal disease model non insulin dependent diabetes mellitus type 2 diabetes mellitus glucokinase knockout mice

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Truncating PICK1 Variant Identified in Azoospermia Affected Mitochondrial Dysfunction in knockout mice

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Current Medical Science 2023年第2期43卷 313-323页

作者： Yao-qiang DU Chong-yi SHU Min ZHENG Wei-de XU Yue SUN Lu SHEN Chen ZHANG Yu-xin ZHANG Qian-ni WANG Kai-qiang LI Bing-yu CHEN Ke HAO Jian-xin LYU Zhen WANG Laboratory Medicine Center Department of Transfusion MedicineZhejiang Provincial People’s HospitalAffiliated People’s HospitalHangzhou Medical CollegeHangzhou 310014China School of Laboratory Medicine and Life Sciences Wenzhou Medical UniversityWenzhou 325035China Center for Reproductive Medicine Department of Reproductive EndocrinologyZhejiang Provincial People’s HospitalAffiliated People’s HospitalHangzhou Medical CollegeHangzhou 310014China Department of Blood Transfusion No.903 Hospital of PLA Joint Logistic Support ForceHangzhou 310000China School of Laboratory Medicine Hangzhou Medical CollegeHangzhou 310059China

Objective The protein interacting with C kinase 1(PICK1)plays a critical role in vesicle trafficking,and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome,which eventually disrupts acrosome formation and leads to male *** An azoospermia sample was filtered,and the laboratory detection and clinical phenotype indicated typical azoospermia in the *** sequenced all of the exons in the PICK1 gene and found that there was a novel homozygous variant in the PICK1 gene,c.364delA(***122SerfsX8),and this protein structure truncating variant seriously affected the biological *** we constructed a PICK1 knockout mouse model using clustered regularly interspaced short palindromic repeat cutting technology(CRISPRc).Results The sperm from PICK1 knockout mice showed acrosome and nucleus abnormalities,as well as dysfunctional mitochondrial sheath *** the total sperm and motility sperm counts were decreased in the PICK1 knockout mice compared to wild-type ***,the mitochondrial dysfunction was verified in the *** defects in the male PICK1 knockout mice may have eventually led to complete *** The c.364delA novel variant in the PICK1 gene associated with clinical infertility,and pathogenic variants in the PICK1 may cause azoospermia or asthenospermia by impairing mitochondrial function in both mice and humans.

关键词： PICK1 azoospermia truncating variant knockout mice mitochondrial dysfunction

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Feeding behavior and gene expression of appetite-related neuropeptides in mice lacking for neuropeptide Y Y5 receptor subclass

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World Journal of Gastroenterology 2008年第41期14卷 6312-6317页

作者： Hiroshi Higuchi Takeshi Niki Tomohiro Shiiya Division of Pharmacology Niigata University Graduate School of Medical and Dental Sciences

Neuropeptide Y (NPY) is a potent neurotransmitter for feeding. Besides NPY, orexigenic neuropeptides such as agouti-related protein (AgRP), and anorexi- genic neuropeptides such as α-melatonin stimulating hormone (MSH) and cocaine-amphetamine-regulated transcript (CART) are also involved in central feeding regulation. During fasting, NPY and AgRP gene expressions are up-regulated and POMC and CART gene ex- pressions are down-regulated in hypothalamus. Based on the network of peptidergic neurons, the former are involved in positive feeding regulation, and the latter are involved in negative feeding, which exert these feeding-regulated peptides especially in paraventricular nucleus (PVN). To clarify the compensatory mecha- nism of knock-out of NPY system on feeding, change in gene expressions of appetite-related neuropeptides and the feeding behavior was studied in NPY Y5-KO mice. Food intake was increased in Y5-KO mice. Fasting increased the amounts of food and water intake in the KO mice more profoundly. These data indicated the compensatory phenomenon of feeding behavior in Y5-KO mice. RT-PCR and ISH suggested that the compensation of feeding is due to change in gene expressions of AgRP, CART and POMC in hypothalamus. Thus, these fi ndings indicated that the compensatory mechanism involves change in POMC/CART gene expression in arcuate nucleus (ARC). The POMC/CART gene expression is important for central compensatory regulation in feeding behavior.

关键词： Neuropeptide Y Y5 receptor Feeding Arcuate nucleus knockout mice

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The Notch signaling pathway in retinal dysplasia and retina vascular homeostasis

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Journal of Genetics and Genomics 2010年第9期37卷 573-582页

作者： Minhua Zheng Zifeng Zhang Xingcheng Zhao Yuqiang Ding Hua Han Department of Medical Genetics and Developmental Biology Fourth Military Medical University Xi'an 710032 China Department of Ophthalmology Xifing Hospital Fourth Military Medical University Xi' an 710032 China Department of Anatomy and Neurobiology Tongji University School of Medicine Shanghai 200092 China

The retina is one of the most essential elements of vision pathway in vertebrate. The dysplasia of retina cause congenital blindness or vision disability in individuals, and the misbalance in adult retinal vascular homeostasis leads to neo adults, such as diabetic retinopathy or age-related macular degeneration. Many developmental signaling pathways are involved in the process of retinal development and vascular homeostasis. Among them, Notch signaling pathway has long been studied, and Notch signaling-interfered mouse models show both neural retina dysplasia and vascular abnormality. In this review, we discuss the roles of Notch signaling in the maintenance of retinal progenitor cells, specification of retinal neurons and glial cells, and the sustaining of retina vascular homeostasis, especially from the aspects of conditional knockout mouse models. The potential of Notch signal manipulation may provide a powerful cell fate- and neovascularization-controUing tool that could have important applications in treatment of retinal diseases.

关键词： Notch signaling retinal dysplasia vascular homeostasis neovascularization knockout mice

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Analysis of the genetic interactions between Cyclin A1, Atm and p53 during spermatogenesis

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Asian Journal of Andrology 2007年第6期9卷 739-750页

作者： Nicole Baiumer Marie-Luise Sandstede Sven Diederichs Gabriele Kohler Carol Readhead Ping Ji Feng Zhang Etmar Bulk Jorg Gromoll Wolfgang E. Berdel Hubert Serve Carsten Muller-Tidow Department of Medicine Hematology and Oncology University of Munster Domagstrasse 3 Munster 48149 Germany Department of Pathology University of Munster Domagstrasse 17 Munster 48149 Germany California Institute of Technology 1200 East California Boulevard Pasadena California 91125 USA Interdisciplinary Center for Clinical Research University of Munster Domagstrasse 3 Munster 48149 Germany Institute of Reproductive Medicine University of Munster Domagstrasse 12 Munster 48129 Germany

Aim： To analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double- strand break repair. Methods： Two lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 （Ccnal）; p53- and Ccnal; Atm-double knockout mice. Results： The block in spermatogenesis observed in Cyclin A1-/- （Ccnal-/-） testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccnal-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccnal-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1. Conclusion： Ccnal-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.

关键词： spermatogenesis testis cell cycle meiosis DNA double-strand break giant cell knockout mice

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Androgen receptor deficiency in monocytes/ macrophages does not alter adiposity or glucose homeostasis in male mice

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Asian Journal of Andrology 2018年第3期20卷 276-283页

作者： Katya B Rubinow Barbara Houston Shari Wang Leela Goodspeed Kayoko Ogimoto Gregory J Morton Christopher McCarty Robert E Braun Stephanie T Page Division of Metabolism Endocrinology and Nutrition Department of Medicine University of Washington School of Medicine Seattle WA 98195 USA Jackson Laboratory Bar Harbor ME 04609 USA

Androgen deprivation in men leads to increased adiposity, but the mechanisms underlying androgen regulation of fat mass have not been fully defined. Androgen receptor （AR） is expressed in monocytes/macrophages, which are resident in key metabolic tissues and influence energy metabolism in surrounding cells. Male mice bearing a cell-specific knockout of the AR in monocytes/macrophages （M-ARKO） were generated to determine whether selective loss of androgen signaling in these cells would lead to altered body composition. Wild-type （WT） and M-ARKO mice （12-22 weeks of age, n = 12 per group） were maintained on a regular chow diet for 8 weeks and then switched to a high-fat diet for 8 additional weeks. At baseline and on both the regular chow and high-fat diets, no differences in lean mass or fat mass were observed between groups. Consistent with the absence of differential body weight or adiposity, no differences in food intake （3.0 ± 0.5 g per day for WT mice vs 2.8 ± 0.4 g per day for M-ARKO mice） or total energy expenditure （0.6 ± 0.1 Kcal h-1 for WT mice vs 0.5 ± 0.1 Kcal h-1 for M-ARKO mice） were evident between groups during high-fat feeding. Liver weight was greater in M-ARKO than that in WT mice （1.5 ± 0.1 g vs 1.3 ± 0.0 g, respectively, P = 0.02）. Finally, M-ARKO mice did not exhibit impairments in glucose tolerance or insulin sensitivity relative to WT mice at any study time point. In aggregate, these findings suggest that AR signaling specifically in monocytes/macrophages does not contribute to the regulation of systemic energy balance, adiposity, or insulin sensitivity in male mice.

关键词： androgen receptor knockout mice macrophages male hypogonadism metabolic syndrome

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CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice

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Asian Journal of Andrology 2022年第3期24卷 266-272页

作者： Yuki Oyama Haruhiko Miyata Keisuke Shimada Yoshitaka Fujihara Keizo Tokuhiro Thomas X Garcia Martin M Matzuk Masahito Ikawa Graduate School of Pharmaceutical Sciences Osaka UniversitySuitaOsaka 565-0871Japan Department of Experimental Genome Research Research Institute for Microbial DiseasesOsaka UniversitySuitaOsaka 565-0871Japan Department of Bioscience and Genetics National Cerebral and Cardiovascular CenterSuitaOsaka 564-8565Japan Department of Genome Editing Institute of Biomedical ScienceKansai Medical UniversityHirakataOsaka 573-1191Japan Center for Drug Discovery Baylor College of MedicineHoustonTX 77030USA Department of Pathology and Immunology Baylor College of MedicineHoustonTX 77030USA Department of Biology and Biotechnology University of Houston-Clear LakeHoustonTX 77058USA The Institute of Medical Science The University of TokyoMinato-kuTokyo 108-8639Japan

Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human *** functional analyses of hundreds of these genes have been performed,there are still many testis-enriched genes whose functions remain *** gene function using knockout(KO)mice is a powerful tool to discern if the gene of interest is essential for sperm formation,function,and male fertility in *** this study,we generated KO mice for 12 testis-enriched genes,1700057G04Rik,4921539E11Rik,4930558C23Rik,Cby2,Ldhal6b,Rasef,Slc25a2,Slc25a41,Smim8,Smim9,Tmem210,and Tomm20l,using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)*** designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null *** tests of KO mice reveal that these 12 genes are not essential for male fertility,at least when individually ablated,and not together with other potentially compensatory paralogous *** results could prevent other laboratories from expending duplicative effort generating KO mice,for which no apparent phenotype exists.

关键词： CRISPR/Cas9 knockout mice male infertility spermatozoa testis

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Potential significance of CX3CR1 dynamics in stress resilience against neuronal disorders

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Neural Regeneration Research 2022年第10期17卷 2153-2156页

作者： Koichi Inoue Department of Integrative Anatomy Nagoya City University Graduate School of Medical SciencesNagoyaJapan

Recent findings have implicated inflammatory responses in the central nervous system in a variety of neuropsychiatric and neurodegenerative diseases,and the understanding and control of immunological responses could be a major factor of future therapeutic strategies for neurological ***,derived from myelogenous cells,respond to a number of stimuli and make immune responses,resulting in a prominent role as cells that act on inflammation in the central nervous ***(FKN or CX3CL1)signaling is an important factor that influences the inflammatory response of *** receptor for FKN,CX3CR1,is usually expressed in microglia in the brain,and therefore the inflammatory response of microglia is modified by ***,FKN often suppresses inflammatory responses in microglia and activation of its receptor may be effective in the treatment of inflammatory neurological ***,it has also been suggested that inflammatory responses facilitated by FKN signaling aggravate neurological ***,further studies are still required to resolve the conflicting interpretation of the protective or deleterious contribution of microglial FKN *** notably,regulation of FKN signaling has recently been shown to be beneficial in the treatment of human diseases,although not neurological *** addition,a CX3CR1 inhibitor has been developed and successfully tested in animal models,and it is expected to be in human clinical trials in the *** this review,I describe the potential therapeutic consideration of microglial CX3CR1 dynamics through altered FKN signaling.

关键词： Alzheimer’s disease CX3CR1 fractalkine inflammation knockout mice microglia resilience SARS-CoV-2 stress stroke

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Osteoblast-restricted Disruption of the Growth Hormone Receptor in mice Results in Sexually Dimorphic Skeletal Phenotypes

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Bone Research 2013年第1期1卷 85-97页

作者： Vandana Singhal Brian C.Goh Mary L.Bouxsein Marie-Claude Faugere Douglas J.DiGirolamo Department of Orthopaedic Surgery Johns Hopkins University School of MedicineBaltimoreMDUSA Center for Advanced Orthopedic Studies Beth Israel Deaconess Medical Center and Harvard Medical SchoolBostonMAUSA Albert B.Chandler Medical Center University of KentuckyLexingtonKYUSA

Growth hormone （GH） exerts profound anabolic actions during postnatal skeletal development, in part, through stimulating the production of insulin-like growth factor-1 （IGF-1） in liver and skeletal tissues. To examine the requirement for the GH receptor （GHR） in osteoblast function in bone, we used Cre-LoxP methods to disrupt the GHR from osteoblasts, both in vitro and in vivo. Disruption of GHR from primary calvarial osteoblasts in vitro abolished GH-induced signaling, as assessed by JAK2/STAT5 phosphorylation, and abrogated GH-induced proliferative and anti-apoptotic actions. Osteoblasts lacking GHR exhibited reduced IGF-l-induced Erk and Akt phosphorylation and attenuated IGF-1-induced proliferation and anti-apoptotic action. In addition, differentiation was modestly impaired in osteoblasts lacking GHR, as demonstrated by reduced alkaline phosphatase staining and calcium deposition. In order to determine the requirement for the GHR in bone in vivo, we generated mice lacking the GHR specifically in osteoblasts （△GHR）, which were born at the expected Mendelian frequency, had a normal life span and were of normal size. Three week-old, female AGHR mice had significantly reduced osteoblast numbers, consistent with the in vitro data. By six weeks of age however, female AGHR mice demonstrated a marked increase in osteoblasts, although mineralization was impaired; a phenotype similar to that observed previously in mice lacking IGF-1R specifically in osteoblasts. The most striking phenotype occurred in male mice however, where disruption of the GHR from osteoblasts resulted in a ＂feminization＂ of bone geometry in 16 week-old mice, as observed by faCT. These results demonstrate that the GHR is required for normal postnatal bone development in both sexes. GH appears to serve a primary function in modulating local IGF-1 action. However, the changes in bone geometry observed in male AGHR mice suggest that, in addition to facilitating IGF-1 action, GH may function to a greater

关键词： growth hormone osteoblasts knockout mice bone sexual dimorphism

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IGF-1 Signaling is Essential for Differentiation of Mesenchymal Stem Cells for Peak Bone Mass

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Bone Research 2013年第2期1卷 186-194页

作者： Janet L. Crane Luo Zhao Joseph S. Frye Lingling Xian Tao Qiu Xu Cao Department of Pediatrics Johns Hopkins University School of Medicine Department of Orthopaedic Surgery Johns Hopkins University School of Medicine Department of Orthopedics Peking Union Medical College HospitalPeking Union Medical College and Chinese Academy of Medical Sciences University of Missouri School of Medicine

Survival of children with chronic medical illnesses is leading to an increase in secondary osteoporosis due to impaired peak bone mass （PBM）. Insulin-like growth factor type Ⅰ （IGF-1） levels correlate with the pattern of bone mass accrual and many chronic illnesses are associated with low IGF-1 levels. Reduced serum levels of IGF-1 minimally affect the integrity of the skeleton, whereas recent studies suggest that skeletal IGF-I regulates PBM. To determine the role of IGF-1 in postnatal bone mass accrual regardless of source, we established an inducible type 1 Igf receptor Cre/lox knockout mouse model, in which the type 1 Igf receptor was deleted inducibely in the mesenchymal stem cells （MSCs） from 3-7 weeks of age. The size of the mouse was not affected as knockout and wild type mice had similar body weights and nasoanal and femoral lengths. However, bone volume and trabecular bone thickness were decreased in the secondary spongiosa of female knockout mice relative to wild type controls, indicating that IGF-1 is critical for bone mass. IGF-1 signaling in MSCs in vitro has been implicated to be involved in both migration to the bone surface and differentiation into bone forming osteoblasts. To clarify the exact role of IGF-1 in bone, we found by immunohistochemical analysis that a similar number of Osterix-positive osteoprogenitors were on the bone perimeter, indicating migration of MSCs was not affected. Most importantly, 56% fewer osteocalcin-positive mature osteoblasts were present on the bone perimeter in the secondary spongiosa in knockout mice versus wild type littermates. These in vivo data demonstrate that the primary role of skeletal IGF-1 is for the terminal differentiation of osteoprogenitors, but refute the role of IGF-1 in MSC migration in vivo. Additionally, these findings confirm that impaired IGF-1 signaling in bone MSCs is sufficient to impair bone mass acquisition.

关键词： osteoblasts knockout mice cell migration IGF receptor nestin

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