• Title/Summary/Keyword: Cell manipulation

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Factors Affecting the Productivity of Germ-line Chimeras from Jl Embryonic Stem Cells (Jl 배아주세포를 이용한 효율적인 생식선 이행 카이미라의 생산)

  • Kim, S.U.;Koo, B.S.;Jeong, S.;Lee, T.H.;Yu, S.L.;Nam, Y.I.;Kim, J.L.;Hyun, B.H.;Shin, H.S.;Lee, K.K.;Sang, B.C.;Yu, D.Y.
    • Korean Journal of Animal Reproduction
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    • v.25 no.1
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    • pp.71-77
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    • 2001
  • This experiment was designed to improve the production efficiency of germ-line chimeric mice from phospholipase C (PLC)-$\beta$3 or peroxiredoxin (Prx) E -targeted ($\Delta$) ES cells by the investigating the manipulation conditions and characteristics of Jl ES cells. Four targeting clones were isolated to investigate the karyotypical and morphological stability prior to injection. All clones ($\Delta$PLC$\beta$-3 C3, $\Delta$Prx II C3, C10 and I5) showed more than 80% euploidism, however, most of $\Delta$PLC$\beta$-3 C3 clones were extensively differentiated compared to the other clones. Nine of 13 $\Delta$Prx II chimeras appeared to have at least 80% chimerism, whereas $\Delta$PLC$\beta$-3 C3 chimeras had 20% chimerism at most. Therefore, the morphological stability of ES cells under stable euploidism might mainly affect the production rate of high-coat chimeric mice. To increase the collection rate of injectable blastocysts (IBs), 5 to 10 week -aged C57BL/6J female mice were sacrificed at 3.5 days post-coitum. Ten week-aged mice were the most optimal IB donors by showing the highest collection rate (2.94/mouse) of injectable blastocysts without increase of non-injectable embryos (0.29/mouse). Foster mothers might be another factor because ICR x C57BL/6J F1 foster mother showed more increased productivity in litter size (2.8 vs. 5.6) and chimera (0 vs. 35.3%) than those of ICR foster mothers. In conclusion, the efficient production of germ-line chimeras mainly depends on the maintenance of ES cell morphology during targeting procedure, and the establishment of manipulation conditions might be a key point to maximize it.

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Construction of hsf1 Knockout-mutant of a Thermotolerant Yeast Strain Saccharomyces cerevisiae KNU5377 (고온내성 연료용 알코올 효모균주 Saccharomyces cerevisiae KNU5377에서 HSF1 유전자의 변이주 구축)

  • Kim Il-Sup;Yun Hae-Sun;Choi Hye-Jin;Sohn Ho-Yong;Yu Choon-Bal;Kim Jong-Guk;Jin Ing-Nyol
    • Journal of Life Science
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    • v.16 no.3 s.76
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    • pp.454-458
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    • 2006
  • HSF1 is the heat shock transcription factor in Saccharomyces cerevisiae. S. cerevisiae KNU5377 can ferment at high temperature such as $40^{\b{o}}C$. We have been the subjects of intense study because Hsf1p mediates gene expression not only to heat shock, but to a variety of cellular and environmental stress challenges. Basing these facts, we firstly tried to construct the hsf1 gene-deleted mutant. PCR-method for fast production of gene disruption cassette was introduced in a thermotolerant yeast S. cerevisiae KNU5377, which allowed the addition of short flanking homology region as short as 45 bp suffice to mediate homologous recombination to kanMX module. Such a cassette is composed of linking genomic DNA of target gene to the selectable marker kanMX4 that confers geneticin (G418) resistance in yeast. That module is extensively used for PCR-based gene replacement of target gene in the laboratory strains. We describe here the generation of hsf1 gene disruption construction using PCR product of selectable marker with primers that provide homology to the hsf1 gene following separation of haploid strain in wild type yeast S. cerevisiae KNU5377. Yeast deletion overview containing replace cassette module, deletion mutant construction and strain confirmation in this study used Saccharomyces Genome Deletion Project (http:://www-sequence.standard.edu/group/yeast_deletion_project). This mutant by genetic manipulation of wild type yeast KNU5377 strain will provide a good system for analyzing the research of the molecular biology underlying their physiology and metabolic process under fermentation and improvement of their fermentative properties.

Production of Supermale (YY) and Superfemale $({\Delta}YY)$ Nile Tilapia (Oreochromis niloticus) by Sex Reversal and Chromosome Manipulation I. Induction of Gynogenetic Diploid from XY Female (성전환 및 염색체 공학 기법을 이용한 초수컷(YY) 및 초암컷$({\Delta}YY)$ 나일틸라피아(Oreochromis niloticus) 생산 I. 성전환된 XY 암컷으로부터 자성발생성 이배체 유도)

  • Kim Dong Soo;Choi Yoon Hee;Noh Choong Hwan;Nam Yoon Kwon
    • Journal of Aquaculture
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    • v.8 no.4
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    • pp.295-306
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    • 1995
  • The present study was performed to produce supermales and superfemales in Oreochromis niloticus by combination of induced sex reversal and diploid gynogenesis. More than $95\%$ of female was obtained by oral administration of $17\beta-estradiol$ (480 mg/kg diet) to the eutheroembryonic larvae of this species. The result of progeny tests with XY pseudofemales showed that incidences of male progeny were ranged from $71.4\%\;to\;73.7\%$. p.opo.lion of male from XY pseudofemale was not significantly different (P>0.05) from that of $\chi^2$ (1:3), but it was significantly different (P<0.01) from that of $\chi^2$(1:1) Gynogenetic diploids were produced by applying cold shocks 3 min after insemination to the eggs of XY pseudofemale sperms were genetically inactivated by ultraviolet rays of 4,050 $erg/mm^2$. Fertilization rates of gynogenetic diploids were not different from that of their controls, however, hatching rates and early survival rates were silghtly lower than those of controls. In the cytogenetic studies, there was no difference in the size of cell and nucleus between the gynogenetic diploids and the controls. At 80 days after hatching, frequencies of gynogenetic male were higher than those of gynogenetic female (P<0.01) in their population.

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Amplification of Porcine SRY Gene for Sex Determination

  • Choi, S.G.;Bae, M.S.;Lee, E.S.;Kim, S.O.;Kim, B.K.;Yang, J.H.;Jeon, C.E.;Kim, H.H.;Hwang, Y.J.;Lee, E.S.;Kim, D.Y.
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.8
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    • pp.1107-1112
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    • 2009
  • The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 $\mu{M}$ Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the Xsorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene.

Effects of the Hippo Signaling Pathway in Human Gastric Cancer

  • Zhou, Guang-Xi;Li, Xiao-Yu;Zhang, Qi;Zhao, Kun;Zhang, Cui-Ping;Xue, Chang-Hu;Yang, Kun;Tian, Zi-Bin
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.9
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    • pp.5199-5205
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    • 2013
  • Background/Aim: The Hippo signaling pathway is a newly discovered and conserved signaling cascade, which regulates organ size control by governing cell proliferation and apoptosis. This study aimed to investigate its effects in human gastric cancer. Methods: Tumor tissues (n=60), adjacent non-tumor tissues (n=60) and normal tissues (n=60) were obtained from the same patients with primary gastric cancer (GC). In addition, 70 samples of chronic atrophic gastritis (CAG) tissues were obtained from patients with intestinal metaplasia (IM) by endoscopic biopsy. Hippo signaling molecules, including Mst1, Lats1, YAP1, TAZ, TEAD1, Oct4 and CDX2, were determined by quantitative polymerase chain reaction (qPCR). Protein expression of Mst1, Lats1, YAP1, TEAD1 and CDX2 was assessed by immunohistochemistry and Western blotting. Results: Mst1, Lats1 and Oct4 mRNA expression showed an increasing tendency from GC tissues to normal gastric tissues, while the mRNA expression of YAP1, TAZ and TEAD1 was up-regulated (all P<0.01). Mst1 and Lats1 protein expression presented a similar trend with their mRNA expression. In addition, YAP1 and TEAD1 protein expression in GC was significantly higher than in the other groups (all P<0.01). CDX2 mRNA and protein expression in the CAG group were higher than in the other groups (all P<0.01). In GC, mRNA expression of Mst1, Lats1, Oct4, YAP1, TAZ, TEAD1 and CDX2 had a close correlation with lymphatic metastasis and tumor TNM stage (all P<0.01). Furthermore, protein expression of Mst1, Lats1, YAP1, TAZ, TEAD1 and CDX2 had a close correlation between each other (P<0.05). Conclusion: The Hippo signaling pathway is involved in the development, progression and metastasis of human gastric cancer. Therefore, manipulation of Hippo signaling molecules may be a potential therapeutic strategy for gastric cancer.

Sarijang Enhances Maturation of Murine Bone Marrow-Derived Dendritic Cells (사리장 처리에 의한 수지상세포의 성숙 유도)

  • Jin, Cheng-Yun;Han, Min-Ho;Park, Cheol;Hwang, Hye-Jin;Choi, Eun-A;Choi, Yung-Hyun
    • Journal of Life Science
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    • v.21 no.12
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    • pp.1789-1794
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    • 2011
  • Dendritic cells (DCs) are professional antigen-presenting cells playing key roles in immune sentinels as initiators of T-cell responses against microbial pathogens and tumors. Sarijang, a folk sauce containing extracts of Rhynchosia nulubilis, Ulmus davidiana roots, Allium sativum, and Rhus Verniaiflura bark, has been used as a nonspecific immunostimulant for cancer patients. However, little is known about its immunomodulating effects or their mechanisms. In this study, we investigated whether sarijang induces phenotypic and functional maturation of DCs. For this study, murine bone marrow-derived myeloid DCs were cultured in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF), and the generated immature DCs were stimulated with sarijang or lipopolysaccharide (LPS). Our data indicated that sarijang significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) as well as major histocompatibility complex (MHC) II, as did LPS. The results provide new insight into the immunopharmacology of sarijang and suggest a novel approach to the manipulation of DC for therapeutic application.

Korean Red Ginseng aqueous extract improves markers of mucociliary clearance by stimulating chloride secretion

  • Cho, Do-Yeon;Skinner, Daniel;Zhang, Shaoyan;Lazrak, Ahmed;Lim, Dong Jin;Weeks, Christopher G.;Banks, Catherine G.;Han, Chang Kyun;Kim, Si-Kwan;Tearney, Guillermo J.;Matalon, Sadis;Rowe, Steven M.;Woodworth, Bradford A.
    • Journal of Ginseng Research
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    • v.45 no.1
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    • pp.66-74
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    • 2021
  • Background: Abnormal chloride (Cl-) transport has a detrimental impact on mucociliary clearance in both cystic fibrosis (CF) and non-CF chronic rhinosinusitis. Ginseng is a medicinal plant noted to have anti-inflammatory and antimicrobial properties. The present study aims to assess the capability of red ginseng aqueous extract (RGAE) to promote transepithelial Cl- secretion in nasal epithelium. Methods: Primary murine nasal septal epithelial (MNSE) [wild-type (WT) and transgenic CFTR-/-], fisher-rat-thyroid (FRT) cells expressing human WT CFTR, and TMEM16A-expressing human embryonic kidney cultures were utilized for the present experiments. Ciliary beat frequency (CBF) and airway surface liquid (ASL) depth measurements were performed using micro-optical coherence tomography (μOCT). Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers and whole-cell patch clamp analysis. Results: RGAE (at 30㎍/mL of ginsenosides) significantly increased Cl- transport [measured as change in short-circuit current (ΔISC = ㎂/㎠)] when compared with control in WT and CFTR-/- MNSE (WT vs control = 49.8±2.6 vs 0.1+/-0.2, CFTR-/- = 33.5±1.5 vs 0.2±0.3, p < 0.0001). In FRT cells, the CFTR-mediated ΔISC attributed to RGAE was small (6.8 ± 2.5 vs control, 0.03 ± 0.01, p < 0.05). In patch clamp, TMEM16A-mediated currents were markedly improved with co-administration of RGAE and uridine 5-triphosphate (8406.3 +/- 807.7 pA) over uridine 5-triphosphate (3524.1 +/- 292.4 pA) or RGAE alone (465.2 +/- 90.7 pA) (p < 0.0001). ASL and CBF were significantly greater with RGAE (6.2+/-0.3 ㎛ vs control, 3.9+/-0.09 ㎛; 10.4+/-0.3 Hz vs control, 7.3 ± 0.2 Hz; p < 0.0001) in MNSE. Conclusion: RGAE augments ASL depth and CBF by stimulating Cl- secretion through CaCC, which suggests therapeutic potential in both CF and non-CF chronic rhinosinusitis.

Phytoplankton Community Change of Lake Paldang by Increasing $CO_2$ and Temperature during Spring Cold Water Season ($CO_2$와 수온 증가에 대한 봄철 저수온기 팔당호 식물플랑크톤군집 변화)

  • Lee, Ka-Ram;Sung, Eun-Ju;Park, Hye-Jin;Park, Chae-Hong;Park, Myung-Hwan;Hwang, Soon-Jin
    • Korean Journal of Ecology and Environment
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    • v.46 no.4
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    • pp.588-595
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    • 2013
  • This study investigated the influence of temperature and $CO_2$ increase on phytoplankton growth and community structure during cold water season (spring) in Lake Paldang, Korea. Four experimental treatments of temperature and $CO_2$ manipulation were prepared in the laboratory batch culture: (1) Control; ambient low temperature ($6{\pm}2^{\circ}C$) and low $CO_2$ (air level, $400mgL^{-1}$), (2) T1; low temperature and high $CO_2$ ($800mgL^{-1}$), (3) T2; high temperature ($20{\pm}2^{\circ}C$) and low $CO_2$, (4) T3; high temperature and high $CO_2$. Algal growth experiment was carried out for 10 days under the light intensity of $70{\mu}mol\;m^{-2}s^{-1}$ (L :D=24 : 0). The level of pH decreased in both T1 and T3, due to dissolution of added $CO_2$. The dominant phytoplankton species of ambient water, Cyclotella meneghiniana succeeded to Fragilaria capucina var. gracilis in high-temperature treatment groups (T2 and T3). Cyanobacteria were very rare at the beginning of the experiment, while Oscillatoria limnetica appeared in only high-temperature groups (T2 and T3) at $6{\sim}7^{th}$ day. $CO_2$ addition in ambient temperature (T1) induced the highest phytoplankton growth, and thereby producing the highest average cell density of $3.27{\pm}0.33\;10^4\;cells\;mL^{-1}$, followed by T2 ($2.65{\pm}0.26\;10^4\;cells\;mL^{-1}$), T3 ($2.09{\pm}0.16\;10^4\;cells\;mL^{-1}$), and Control ($1.86{\pm}0.13\;10^4\;cells\;mL^{-1}$) (F=7.167, p=0.000). In summary, temperature increase changed the phytoplankton community structure and $CO_2$ increase promoted the phytoplankton growth during the cold spring season in Lake Paldang, suggesting a potential effect of climate change on freshwater phytoplankton.

The Effect of Price Promotional Information about Brand on Consumer's Quality Perception: Conditioning on Pretrial Brand (품패개격촉소신식대소비자질량인지적영향(品牌价格促销信息对消费者质量认知的影响))

  • Lee, Min-Hoon;Lim, Hang-Seop
    • Journal of Global Scholars of Marketing Science
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    • v.19 no.3
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    • pp.17-27
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    • 2009
  • Price promotion typically reduces the price for a given quantity or increases the quantity available at the same price, thereby enhancing value and creating an economic incentive to purchase. It often is used to encourage product or service trial among nonusers of products or services. Thus, it is important to understand the effects of price promotions on quality perception made by consumer who do not have prior experience with the promoted brand. However, if consumers associate a price promotion itself with inferior brand quality, the promotion may not achieve the sales increase the economic incentives otherwise might have produced. More specifically, low qualitative perception through price promotion will undercut the economic and psychological incentives and reduce the likelihood of purchase. Thus, it is important for marketers to understand how price promotional informations about a brand have impact on consumer's unfavorable quality perception of the brand. Previous literatures on the effects of price promotions on quality perception reveal inconsistent explanations. Some focused on the unfavorable effect of price promotion on consumer's perception. But others showed that price promotions didn't raise unfavorable perception on the brand. Prior researches found these inconsistent results related to the timing of the price promotion's exposure and quality evaluation relative to trial. And, whether the consumer has been experienced with the product promotions in the past or not may moderate the effects. A few studies considered differences among product categories as fundamental factors. The purpose of this research is to investigate the effect of price promotional informations on consumer's unfavorable quality perception under the different conditions. The author controlled the timing of the promotional exposure and varied past promotional patterns and information presenting patterns. Unlike previous researches, the author examined the effects of price promotions setting limit to pretrial situation by controlling potentially moderating effects of prior personal experience with the brand. This manipulations enable to resolve possible controversies in relation to this issue. And this manipulation is meaningful for the work sector. Price promotion is not only used to target existing consumers but also to encourage product or service trial among nonusers of products or services. Thus, it is important for marketers to understand how price promotional informations about a brand have impact on consumer's unfavorable quality perception of the brand. If consumers associate a price promotion itself with inferior quality about unused brand, the promotion may not achieve the sales increase the economic incentives otherwise might have produced. In addition, if the price promotion ends, the consumer that have purchased that certain brand will likely to display sharply decreased repurchasing behavior. Through a literature review, hypothesis 1 was set as follows to investigate the adjustive effect of past price promotion on quality perception made by consumers; The influence that price promotion of unused brand have on quality perception made by consumers will be adjusted by past price promotion activity of the brand. In other words, a price promotion of an unused brand that have not done a price promotion in the past will have a unfavorable effect on quality perception made by consumer. Hypothesis 2-1 was set as follows : When an unused brand undertakes price promotion for the first time, the information presenting pattern of price promotion will have an effect on the consumer's attribution for the cause of the price promotion. Hypothesis 2-2 was set as follows : The more consumer dispositionally attribute the cause of price promotion, the more unfavorable the quality perception made by consumer will be. Through test 1, the subjects were given a brief explanation of the product and the brand before they were provided with a $2{\times}2$ factorial design that has 4 patterns of price promotion (presence or absence of past price promotion * presence or absence of current price promotion) and the explanation describing the price promotion pattern of each cell. Then the perceived quality of imaginary brand WAVEX was evaluated in the scale of 7. The reason tennis racket was chosen is because the selected product group must have had almost no past price promotions to eliminate the influence of average frequency of promotion on the value of price promotional information as Raghubir and Corfman (1999) pointed out. Test 2 was also carried out on students of the same management faculty of test 1 with tennis racket as the product group. As with test 1, subjects with average familiarity for the product group and low familiarity for the brand was selected. Each subjects were assigned to one of the two cells representing two different information presenting patterns of price promotion of WAVEX (case where the reason behind price promotion was provided/case where the reason behind price promotion was not provided). Subjects looked at each promotional information before evaluating the perceived quality of the brand WAVEX in the scale of 7. The effect of price promotion for unfamiliar pretrial brand on consumer's perceived quality was proved to be moderated with the presence or absence of past price promotion. The consistency with past promotional behavior is important variable that makes unfavorable effect on brand evaluations get worse. If the price promotion for the brand has never been carried out before, price promotion activity may have more unfavorable effects on consumer's quality perception. Second, when the price promotion of unfamiliar pretrial brand was executed for the first time, presenting method of informations has impact on consumer's attribution for the cause of firm's promotion. And the unfavorable effect of quality perception is higher when the consumer does dispositional attribution comparing with situational attribution. Unlike the previous studies where the main focus was the absence or presence of favorable or unfavorable motivation from situational/dispositional attribution, the focus of this study was exaus ing the fact that a situational attribution can be inferred even if the consumer employs a dispositional attribution on the price promotional behavior, if the company provides a persuasive reason. Such approach, in academic perspectih sis a large significance in that it explained the anchoring and adjng ch approcedures by applying it to a non-mathematical problem unlike the previous studies where it wis ionaly explained by applying it to a mathematical problem. In other wordn, there is a highrspedency tmatispositionally attribute other's behaviors according to the fuedach aal attribution errors and when this is applied to the situation of price promotions, we can infer that consumers are likely tmatispositionally attribute the company's price promotion behaviors. Ha ever, even ueder these circumstances, the company can adjng the consumer's anchoring tmareduce the po wibiliute thdispositional attribution. Furthermore, unlike majority of previous researches on short/long-term effects of price promotion that only considered the effect of price promotions on consumer's purchasing behaviors, this research measured the effect on perceived quality, one of man elements that affects the purchasing behavior of consumers. These results carry useful implications for the work sector. A guideline of effectively providing promotional informations for a new brand can be suggested through the outcomes of this research. If the brand is to avoid false implications such as inferior quality while implementing a price promotion strategy, it must provide a clear and acceptable reasons behind the promotion. Especially it is more important for the company with no past price promotion to provide a clear reason. An inconsistent behavior can be the cause of consumer's distrust and anxiety. This is also one of the most important factor of risk of endless price wars. Price promotions without prior notice can buy doubt from consumers not market share.

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