• Title, Summary, Keyword: Knockout

Search Result 293, Processing Time 0.049 seconds

Epinephrine Control of Glycogen Metabolism in Glycogen-associated Protein Phosphatase PP1G/RGLKnockout Mice

  • 김종화;Anna A. DePaoli-Roach
    • BMB Reports
    • /
    • v.35 no.3
    • /
    • pp.283-290
    • /
    • 2002
  • The glycogen-associated protein phosphatase (PP1G/$R_{GL}$) may play a central role in the hormonal control of glycogen metabolism in the skeletal muscle. Here, we investigated the in vivo epinephrine effect of glycogen metabolism in the skeletal muscle of the wild-type and $R_{GL}$ knockout mice. The administration of epinephrine increased blood glucose levels from 200±20 to 325±20 mg/dl in both wild-type and knockout mice. Epinephrine decreased the glycogen synthase -/+ G6P ratio from 0.24±0.04 to 0.10±0.02 in the wild-type, and from 0.17±0.02 to 0.06±0.01 in the knockout mice. Conversely, the glycogen phosphorylase activity ratio increased from 0.21±0.04 to 0.65±0.07 and from 0.30±0.04 to 0.81±0.06 in the epinephrine trated wild-type and knockout mice respectively. The glycogen content of the knockout mice was substantially lower (27%) than that of both wild-type mice; and epinephrine decreased glycogen content in the wild-type and knockout mice. Also, in Western blot analysis there was no compensation of the other glycogen targeting components PTG/R5 and R6 in the knockout mice compared with the wild-type. Therefore, $R_{GL}$ is not required for the epinephrine stimulation of glycogen metabolism, and rather another phosphatase and/or regulatory subunit appears to be involved.

Development of Reusable Split URA3-Marked Knockout Vectors for Saccharomyces cerevisiae

  • Lee Tae-Hee;Kim Myoung-Dong;Seo Jin-Ho
    • Journal of Microbiology and Biotechnology
    • /
    • v.16 no.6
    • /
    • pp.979-982
    • /
    • 2006
  • Two knockout vectors, in which the truncated Kluyveromyces lactis URAS gene is flanked by a direct repeat, were developed for Saccharomyces cerevisiae. Each vector was designed to harbor 5'- and 3'-end homology regions for integration. Two knockout fragments were devised to integrate into the correct locus in a complementary manner to disrupt a gene of interest and. concomitantly to make functional Kl URA3 for transfomant selection. The use of dual complementary knockout cassettes was expected to dramatically reduce integration into unwanted loci in the genome. The knockout system developed in this study was successfully used for disruption of the GAL1 gene in S. cerevisiae.

Development of Cre-lox based multiple knockout system in Deinococcus radiodurans R1

  • Jeong, Sun-Wook;Yang, Jung Eun;Im, Seonghun;Choi, Yong Jun
    • The Korean Journal of Chemical Engineering
    • /
    • v.34 no.6
    • /
    • pp.1728-1733
    • /
    • 2017
  • The extremophilic bacterium Deinococcus radiodurans R1 has been considered as an attractive microorganism due to its remarkable tolerance to various external stresses. Considering the nature of D. radiodurans R1, it has potential as a platform microorganism for industrial applications, including biorefinery and bioremediation process. However, D. radiodurans R1 is well known for its hard genetic manipulation. Thus, much effort has been made to develop efficient genetic engineering tools for making D. radiodurans R1 suitable for industrial platform microorganism. Although a plasmid-based single gene knockout method has been reported, development of multiple gene knockout system has not yet been reported. Here we report, for the first time, Cre-lox based rapid and efficient multiple knockout method for metabolic engineering of D. radiodurans R1. Also, deletion of dr0053 gene was successfully achieved within seven days to make biofilm overproducing strain.

BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

  • Hei, Hongya;Gao, Jianjun;Dong, Jibin;Tao, Jie;Tian, Lulu;Pan, Wanma;Wang, Hongyu;Zhang, Xuemei
    • Molecules and Cells
    • /
    • v.39 no.7
    • /
    • pp.530-535
    • /
    • 2016
  • Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BKknockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect

  • Lee, Jeong Hyo;Kim, Si Won;Park, Tae Sub
    • Asian-Australasian Journal of Animal Sciences
    • /
    • v.30 no.5
    • /
    • pp.743-748
    • /
    • 2017
  • Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results: Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion: The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

Production of Knockout Mice using CRISPR/Cas9 in FVB Strain

  • Bae, Hee Sook;Lee, Soo Jin;Koo, Ok Jae
    • Journal of Embryo Transfer
    • /
    • v.30 no.4
    • /
    • pp.299-303
    • /
    • 2015
  • KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing

  • Park, Bo Min;Roh, Jae-il;Lee, Jaehoon;Lee, Han-Woong
    • Laboraroty Animal Research
    • /
    • v.34 no.4
    • /
    • pp.264-269
    • /
    • 2018
  • Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors $p16^{Ink4a}$ (Cdkn2a, cyclin-dependent kinase inhibitor 2a), $p19^{Arf}$ (an alternative reading frame product of Cdkn2a,), and $p27^{Kip1}$ (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The $p16^{Ink4a}$ and $p19^{Arf}$ knockout mice were generated via transcription activator-like effector nucleases (TALENs), and $p27^{Kip1}$ knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

Infection with Citrobacter rodentium in μMT Knockout Mice

  • Jo, Minjeong;Hwang, Soonjae;Rhee, Ki-Jong
    • Biomedical Science Letters
    • /
    • v.24 no.1
    • /
    • pp.1-8
    • /
    • 2018
  • ${\mu}MT$ knockout mice are genetically deficient in the transmembrane domain of mu chain of the immunoglobulin M (IgM) heavy chain, resulting in the absence of mature B cells. ${\mu}MT$ knockout mice is an in vivo model system used to clarify the role of B cells in various diseases. Enteropathogenic Escherichia coli (EPEC) induces acute and chronic diarrheal disease, especially in children of developing countries. The formation of attaching and effacing (A/E) lesion is a prominent pathogenic factor in the intestinal epithelium of EPEC infection. The A/E lesion is modulated by genes located on the pathogenic island locus of enterocyte effacement (LEE) which encode a type III secretion system (T3SS) and A/E lesion-related effector proteins. Citrobacter rodentium is a murine pathogen utilized in studying the pathogenic mechanisms of EPEC in human infections. Citrobacter rodentium produce A/E lesion to attach to intestinal epithelium, thus providing a murine model pathogen to study EPEC. Several studies have investigated the pathogenesis of Citrobacter rodentium in the ${\mu}MT$ knockout mice. In this review, we introduce the ${\mu}MT$ murine model in the context of C. rodentium pathogenesis and describe in detail the role of B cells and antibodies in this disease.

Performance evaluation of the input and output buffered knockout switch

  • Suh, Jae-Joon;Jun, Chi-Hyuck;Kim, Young-Si
    • Korean Management Science Review
    • /
    • v.10 no.1
    • /
    • pp.139-156
    • /
    • 1993
  • Various ATM switches have been proposed since Asynchronous Transfer Mode (ATM) was recognized as appropriate for implementing broadband integrated services digital network (BISDN). An ATM switching network may be evaluated on two sides : traffic performances (maximum throughput, delay, and packet loss probability, etc.) and structural features (complexity, i.e. the number of switch elements necessary to construct the same size switching network, maintenance, modularity, and fault tolerance, etc.). ATM switching networks proposed to date tend to show the contrary characteristics between structural features and traffic performance. The Knockout Switch, which is well known as one of ATM switches, shows a good traffic performance but it needs so many switch elements and buffers. In this paper, we propose an input and output buffered Knockout Switch for the purpose of reducing the number of switch elements and buffers of the existing Knockout Switch. We analyze the traffic performance and the structural features of the proposed switching architecture through a discrete time Markov chain and compare with those of the existing Knockout Switch. It was found that the proposed architecture could reduce more than 40 percent of switch elements and more than 30 percent of buffers under a given requirement of cell loss probability of the switch.

  • PDF