• Title, Summary, Keyword: Knock-out

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Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus

  • Kim, Ji-Woo;Kim, Hye-Min;Lee, Sang-Mi;Kang, Man-Jong
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.10
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    • pp.1473-1480
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    • 2012
  • The Galactose-${\alpha}1$,3-galactose (${\alpha}1$,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of ${\alpha}1$,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Effect of the pat, fk, stpk Gene Knock-out and mdh Gene Knock-in on Mannitol Production in Leuconostoc mesenteroides

  • Peng, Yu-Wei;Jin, Hong-Xing
    • Journal of Microbiology and Biotechnology
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    • v.28 no.12
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    • pp.2009-2018
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    • 2018
  • Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study we modified the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains for mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deletion strain (${\Delta}pat::amy$), the fk gene (which encodes fructokinase) deletion strain (${\Delta}fk::amy$) and the stpk gene (which encodes serine-threonine protein kinase) deletion strain (${\Delta}stpk::amy$) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1%, respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (${\Delta}pat::mdh$, ${\Delta}fk::mdh$ and ${\Delta}stpk::mdh$) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ${\Delta}dts{\Delta}ldh{\Delta}pat::mdh{\Delta}stpk::mdh{\Delta}fk::mdh$ had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.

A study on the development of temperature and pressure at the end-gas zone during the combustion period to establish the knock theory (노크이론 확립을 위한 말단가스 온도 및 압력 경과이력)

  • 이성열;오영일
    • Journal of the korean Society of Automotive Engineers
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    • v.15 no.1
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    • pp.28-36
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    • 1993
  • Present-day there are two of theories which have considerable scientific support to explain the knock phenomenon in S.I. engine, the detonation theory and the autoignition theory. But they still have some problems to explain effects of knock parameters, i.e.. compression ratio, spark timing, mixture quality, engine speed, ect, on knocking process in S.I. engine. Accordingly, it is essential to find out whish is more adequate theory of two and to develop the method of analyzing knock phenomenon, that is the aim of this paper. The Authors develop the method of predicting transient temperature and pressure at the end-gas zone during the combustion period and analyze knocking process by this method based on the knock theories. The caluculated values based on the autoignition theory show reasonablly correct relations between knock parameters and knock process but there is no evidence of knock occurred by detonation theory through the calculation according to the all parameters. The authors find out that the autoignition theory is more adequate than detonation theory to analyze knocking process in S.I. engine.

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Measurement and Analysis of Knock for Rapid Throttle Opening in SI Engines (가솔린 엔진에서 급가속 운전시 노킹 측정 및 분석)

  • 이종화;박경석;김현용
    • Transactions of the Korean Society of Automotive Engineers
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    • v.7 no.9
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    • pp.28-35
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    • 1999
  • In this study, investigation of transient knock characteristics in a spark-ignition engine has been carried out. The universal knock threshold values were found by a DFDD method and a NSDBP method which is a non-dimensional version of the SDBP method. Also modified NSDBP method could be used for transient knock detection. In a commercial ECU , spark timing was retarded from the steady -state spark timing during rapid throttle opening to avoid uncomfortable feeling and knock. Knock usually occurred just after the start of rapid throttle opening when spark timing was set, as values for the steady state condition. We found that air/fuel ratio deeply involved with the knock during transient condition. Due to the difference of initial heat release rate, knock occurred more easily at rich air/fuel ratio than at lean air/fuel ratio.

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Measurement and Analysis of Knock Using a Microphone Sensor in a S.I. Engine (전기점화기관에서 마이크로폰 센서를 이용한 노킹 측정 및 분석)

  • 황승환;이종화;임진수
    • Transactions of the Korean Society of Automotive Engineers
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    • v.5 no.3
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    • pp.202-208
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    • 1997
  • The knocking is one of major parameters to improve engine performance in a spark ignition engine. Many researches have been carried out to identify them using cylinder pressure, vibration signal and so on. In the present study, measurement and analysis was conducted to set up the criteria of knock occurrence by using microphone signal. Cylinder pressure was measured for the reference signal of knocking. It has been observed that resonance frequencies of pressure wave are nearly independent of engine operating conditions such as engine speed, air fuel ratio, load and octane number of fuel within to limited experimental conditions. SDBP(sum of different band-pass data) method using resonance frequency of knock was proposed for estimating knock intensity. SDBP method is superior to identify knock occurrence and its intensity in case of sound pressure measurement.

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Recent Progress in Biotechnology-based Gene Manipulating Systems to Produce Knock-In/Out Mouse Models

  • Lee, Woon Kyu;Park, Joong Jean;Cha, Seok Ho;Yun, Cheol-Heui
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.5
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    • pp.745-753
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    • 2008
  • Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

Ectopic Expression of Cenexin1 S796A Mutant in $ODF2^{+/-}$ Knockout Background Causes a Sperm Tail Development Defect

  • Lee, Kyung Ho
    • Development and Reproduction
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    • v.16 no.4
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    • pp.363-370
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    • 2012
  • The outer dense fiber 2 (ODF2) protein is an important component of sperm tail outer dense fiber and localizes at the centrosome. It has been reported that the RO072 ES cell derived homozygote knock out of ODF2 results in an embryonic lethal phenotype, and XL169 ES cell derived heterozygote knock out causes severe defects in sperm tail development. The ODF2s splicing variant, Cenexin1, possesses a C-terminal extension, and the phosphorylation of serine 796 residue in an extended C-terminal is responsible for Plk1 binding. Cenexin1 assembles ninein and causes ciliogenesis in early stages of the cell cycle in a Plk1-independent manner. Alternatively, in the late stages of the cell cycle, G2/M phase, Cenexin1 binds to Plk1 and results in proper mitotic progression. In this study, to identify the in vivo function of Plk1 binding to phosphorylated Cenexin1 S796 residue, and to understand the in vivo functional differences between ODF2 and Cenexin1, we generated ODF2/Cenexin1 S796A/Cenexin1 WT expressing transgenic mice in a RO072 ES cell derived $ODF2^{+/-}$ knock out background. We observed a severe defect of sperm tail development by ectopic expression of Cenexin1 S796A mutant and no phenotypic differences between the ectopic expression of ODF2/Cenexin1 WT in $ODF2^{+/-}$ background and in normal wild type mice.

Construction and Expression Analysis of Knock-in Vector for EGFP Expression in the Porcine $\beta$-Casein Gene Locus (돼지 $\beta$-Casein을 이용한 EGFP 발현 Knock-in 벡터의 구축 및 발현 검증)

  • Lee, Sang-Mi;Kim, Hey-Min;Moon, Seung-Ju;Kang, Man-Jong
    • Reproductive and Developmental Biology
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    • v.32 no.3
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    • pp.205-209
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    • 2008
  • This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene ($neo^{r}$) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

Physiological Roles of Bacillus subtilis thiol peroxidase gene in response to oxidative stress (산화적 스트레스에 대한 Bacillus subtilis의 thiol peroxidase 유전자의 생리적인 기능)

  • Kim, Ha-Kun;Kim, Sung-Jin
    • The Journal of Natural Sciences
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    • v.15 no.1
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    • pp.57-67
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    • 2005
  • In Order to investigate the physiological role of thiol peroxidase in Bacillus subtilis, a thiol peroxidase (btpx) knock-out mutant was generated by homologous recombination. The growth of btpx knock-out mutant in aerobic condition showed a similar pattern with that of wild type of Bacillus subtilis 168/ But btpx knock-out mutant showed a retarded growht in response to oxidative stress such as $H_2O_2$, cumene hydroperoxide (CHP) treatments.

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Reverse-engineering of Gene Regulatory Network of S. cerevisiae using Knock-out Data (Knock-out Data 를 이용한 S. Cerevisiae 유전자 조절망의 재구성)

  • Hong, Seong-Yong;Sohn, Ki-Rack
    • Proceedings of the Korea Information Processing Society Conference
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    • pp.603-606
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    • 2005
  • 하나의 유전자는 또 다른 유전자의 단백질과 프로모터 영역에서 Binding 함으로써 그 유전자의 발현에 영향을 미칠 수 있다. 이러한 두 유전자간의 조절 상호 작용을 유전자 조절망이라 하며 유전체의 핵심적인 기능을 보다 간결하게 표현하는 조절망을 설계할 수 있다. 대표적인 설계 방법으로는 Time-Series Data 를 이용한 방법과 Steady-State Data 를 이용하는 방법이 있으며 이 논문에서는 Steady-State Data 즉, Knock-out Data 를 이용하여 유전자 조절망을 재구성함으로써 기존의 방법을 개선하여 보다 정확한 결과 예측을 목표로 한다.

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