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Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein(RNP)electroporation

Journal of Animal Science and Biotechnology 2024年第1期15卷 456-462页

作者： Gyeong-Min Gim Kyeong-Hyeon Eom Dong-Hyeok Kwon Dae-Jin Jung Dae-Hyun Kim Jun-Koo Yi Jae-Jung Ha Ji-Hyun Lee Seong-Beom Lee Woo-Jae Son Soo-Young Yum Won-Wu Lee Goo Jang Laboratory of Theriogenology and Biotechnology Department of Veterinary Clinical ScienceCollege of Veterinary Medicine and the Research Institute of Veterinary ScienceSeoul National UniversitySeoulRepublic of Korea LARTBio Co. Ltd.SeoulRepublic of Korea Gyeongsangbukdo Livestock Research Institute YeongjuRepublic of Korea Comparative Medicine Disease Research Center Seoul National UniversitySeoulRepublic of Korea

Background Genome editing has been considered as powerful tool in agricultural ***,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising ***,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in *** this point of view,electroporation in embryos has been risen as an *** First,editing efficiency of our electroporation methods were tested for *** of mutation on embryo was confirmed by T7E1 *** first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,*** case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for ***,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep *** recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double *** surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both *** These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively ***,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

关键词： Beta-lactoglobulin Cattle CRISPR-Cas9 Electroporation knockout MSTN PRNP

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Neuropeptide Y receptor Y8b(npy8br)regulates feeding and digestion in Japanese medaka(Oryzias latipes)larvae:evidence from gene knockout

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2024年第7期25卷 605-616页

作者： Xiaodan JIA Ke LU Xufang LIANG College of Fisheries Chinese Perch Research CenterHuazhong Agricultural UniversityWuhan 430070China Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt Ministry of EducationWuhan 430070China

Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and *** in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at *** order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their ***,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly *** deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal *** far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

关键词： Neuropeptide Y receptor Y8b(npy8br) Japanese medaka(Oryzias latipes) knockout Feeding Digestion

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knockout of lipoprotein lipase with CRISPR/Cas9 causes severe developmental defects and affects lipid deposition in Japanese medaka(Oryzias latipes)

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Water Biology and Security 2022年第2期1卷 105-111页

作者： Hexiong Feng Xu-Fang Liang College of Fisheries and Chinese Perch Research Center Huazhong Agricultural UniversityWuhan430070China Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt Ministry of EducationWuhan430070China

In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand *** deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause *** lpl gene is not expressed in adult mammalian liver,but is in adult fish *** functions provided by the lpl gene are diverse in ***,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 *** lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the *** is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the *** lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in *** lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in *** general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.

关键词： Lipoprotein lipase knockout Lipid Japanese medaka(Oryzias latipes)

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Fidgetin knockdown and knockout influences female reproduction distinctly in mice

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The Journal of Biomedical Research 2022年第4期36卷 269-279页

作者： Cong-Rong Li Ruo-Lei Wang Shi-Ya Xie Yan-Ru Li Lei-Lei Gao Zhi-Xia Yang Dong Zhang State Key Lab of Reproductive Medicine Nanjing Medical UniversityNanjingJiangsu 211166China Animal Core Facility Nanjing Medical UniversityNanjingJiangsu 211166China

Microtubule-severing proteins(MTSPs),are a family of proteins which use adenosine triphosphate to sever *** have been shown to play an important role in multiple microtubule-involved cellular *** member of this family,fidgetin(FIGN),is also involved in male fertility;however,no studies have explored its roles in female *** this study,we found mouse fidgetin is rich within oocyte zona pellucida(ZP)and is the only MTSP member to do *** also appears to interact with all three ZP *** findings prompted us to propose that fidgetin might prevent *** from in vitro maturation oocytes analysis showed that fidgetin knockdown did cause *** then deleted all three fidgetin isoforms with CRISPR/Cas9 technologies;however,female mice remained healthy and with normal *** all mouse MTSPs,only the mRNA level of fidgetin-like 1(FIGNL1)significantly ***,we assert that fidgetin-like 1 compensates fidgetin's roles in fidgetin knockout female mice.

关键词： mouse fidgetin knockdown knockout female reproduction

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knockout reaction mechanism studied by ~6He projectile

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Science China(Physics,Mechanics & Astronomy) 2011年第S1期54卷 136-140页

作者： Lü LinHui1, YE YanLin1, JIANG DongXing1, HUA Hui1, ZHENG Tao1, LI ZhiHuan1, GE YuCheng1, LI XiangQing1, LOU JianLing1, CAO ZhongXin1, SONG YuShou1, XIAO Jun1, LI QiTe1, QIAO Rui1, YOU HaiBo1, CHEN RuiJiu1, XU HuShan2, WANG JianSong2, GUO ZhongYan2, ZHANG XueYing2, LI Chen2, HU ZhengGuo2, CHEN RuoFu2, WANG Meng2, XU ZhiGuo2, YUE Ke2, TANG Bin2, ZANG YongDong2, ZHANG XueHeng2, YAO XiangWu2, CHEN JinDa2 & BAI Zhen2 1 School of Physics and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871, China 2 Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China School of Physics and State Key Laboratory of Nuclear Physics and Technology Peking University Beijing China Institute of Modern Physics Chinese Academy of Sciences Lanzhou China

knockout reaction experiment was carried out by using the 6He beams at 61.2 MeV/u impinging on a CH2 target. The α core fragments at forward angles were detected in coincidence with the recoiled protons at larger angles. From this exclusive meas urement the valence nucleon knockout mechanism and the core knockout mechanism can be distinguished by the relation be tween the polar angles of the core fragments and the recoiled protons, respectively. It is demonstrated that the core knockout mechanism may result in some strong contamination to the real invariant mass spectrum.

关键词： exotic nuclei knockout reaction reaction mechanism invariant mass spectra

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Reduced prostate branching morphogenes,s in stromal fibroblast, but not in epithelial, estrogen receptor a knockout mice

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Asian Journal of Andrology 2012年第4期14卷 546-555页

作者： Ming Chen Chiuan-Ren Yeh Chih-Rong Shyr Hsiu-Hsia Lin Iun Da Shuyuan Yeh Departments of Urology and Pathology University of Rochester Medical Center Rochester New York 14642 USA Sex Hormone Research Center China Medical University Hospital Taichung 40402 Taiwan China

Early studies suggested that estrogen receptor alpha （ERa） is involved in estrogen-mediated imprinting effects in prostate development. We recently reported a more complete ERa knockout （KO） mouse model via mating β-actin Cre transgenic mice with floxed ERa mice. These ACTB-ERaKO male mice showed defects in prostatic branching morphogenesis, which demonstrates that ERa is necessary to maintain proliferative events in the prostate. However, within which prostate cell type ERa exerts those important functions remains to be elucidated. To address this, we have bred floxed ERa mice with either fibroblast-specific protein （FSP）-Cre or probasin-Cre transgenic mice to generate a mouse model that has deleted ERa gene in either stromal fibroblast （FSP-ERaKO） or epithelial （pes-ERaKO） prostate cells. We found that circulating testosterone and fertility were not altered in *** and pes-ERaKO male mice. Prostates of FSP-ERaKO mice have less branching morphogenesis compared to that of wild-type littermates. Further analyses indicated that loss of stromal ERa leads to increased stromal apoptosis, reduced expression of insulin-likegrowth factor-1 （IGF-1） and FGFIO, and increased expression of BMP4. Collectively, we have established the first in vivo prostate stromal and epithelial selective ERaKO mouse models and the results from these mice indicated that stromal fibroblast ERa plays important roles in prostatic branching morphogenesis via a paracrine fashion. Selective deletion of the ERa gene in mouse prostate epithelial cells by probasin-Cre does not affect the regular prostate development and homeostasis.

关键词： Cre-loxP estrogen receptor knockout prostate stromal-epithelial interaction

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CRISPR Primer Designer:Design primers for knockout and chromosome imaging CRISPR-Cas system

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Journal of Integrative Plant Biology 2015年第7期57卷 613-617页

作者： Meng Yan Shi-Rong Zhou Hong-Wei Xue National Key Laboratory of Plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecologythe Chinese Academy of Sciences

The clustered regularly interspaced short palin- dromic repeats （CRiSPR）-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user- friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present researchers to design primers CRISPR Primer Designer for for CRISPR applications. Theprogram has a user-frfendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRiSPR-Cas system-based chromosome imaging system.

关键词： Chromosome imaging CRISPR knockout primer software

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Uses of knockout,knockdown,and transgenic models in the studies of glucose transporter 4

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World Journal of Meta-Analysis 2022年第1期10卷 1-11页

作者： Tian-Nan Wang Xin-Ge Hu Guo-Xun Chen Department of Nutrition The University of TennesseeKnoxvilleTN 37996United States

Currently,glucose transporter 4(GLUT4)has been considered as the key player for the insulin-stimulated glucose transport in the muscle and adipose *** development of recombinant DNA techniques allows the creations of genetically knockout,knockdown and transgenic animals and cells for the study of GLUT4’s physiological ***,we have used key words to search the PubMed and summarized the methods used in Slc2a4 gene knockout,GLUT4 knockdown and overexpression in the whole body and tissue specific *** whole body GLUT4-null mice have growth retardation,but normal glucose tolerance and basal glucose turnover *** with whole body Slc2a4 knockout mice,adipose and muscle double knockout mice have impaired insulin tolerance and glucose *** results of GLUT4 knockdown in 3T3-L1 adipocytes have shown that its expression is needed for lipogenesis after,but not during,*** mice with the whole body GLUT4 overexpression have normal body weight and lowered blood glucose *** adipose tissue specific overexpression of GLUT4 leads to increases in mouse body weight and adipose tissue *** insulin-stimulated GLUT4 translocation in the skeletal muscle contributes to the regulation of glucose *** from both transgenic overexpression and tissue specific Slc2a4 knockout indicate that GLUT4 probably plays a role in the glucose uptake in the fasting *** studies are warranted to use advanced molecular biology tools to decipher the roles of GLUT4 in the control of glucose homeostasis.

关键词： Glucose transporter 4 knockout Knockdown Transgene Overexpression Insulin

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Claudin-7 gene knockout causes destruction of intestinal structure and animal death in mice

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World Journal of Gastroenterology 2019年第5期25卷 584-599页

作者： Chang Xu Kun Wang Yu-Han Ding Wen-Jing Li Lei Ding Department of Oncology Beijing Shijitan Hospital Capital Medical University

BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP *** To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout *** We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation *** We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were als

关键词： Claudin-7 Gene knockout Inflammation Adenomas Colorectal carcinoma

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Targeted knockout of duox causes defects in zebrafish growth, thyroid development, and social interaction

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Journal of Genetics and Genomics 2019年第2期46卷 101-104页

作者： Jong-Su Park Tae-Ik Choi Oc-Hee Kim Ted Inpyo Hong Woo-Keun Kim Won-Jae Lee Cheol-Hee Kim Department of Biology Chungnam National University Biosystem Research Group Korea Institute of Toxicology School of Biological Sciences Seoul National University

Reactive oxygen species(ROS) are believed to be inevitable and toxic by-products produced during cellular respiration. However,several lines of evidence show that diverse extracellular signals can activate ROS product... 详细信息

关键词： KO In Targeted knockout of duox causes defects in zebrafish growth thyroid development and social interaction

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