• Title, Summary, Keyword: stable transfection

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New Cationic Liposome with Enhanced Stability and Transfection Efficiency for Gene Delivery (안정성 및 Transfection 효율이 우수한 양이온성 리포좀 유전자 전달시스템의 개발)

  • Kim, Kyoung-Mi;Nam, Bang-Hyun;Sohn, Dong-Hwan
    • Journal of Pharmaceutical Investigation
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    • v.28 no.2
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    • pp.93-98
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    • 1998
  • We have developed liposomes which can be easily prepared with inexpensive lipid, have enhanced stability, and can efficiently deliver DNA into the COS-l cells, Liposome formulations were prepared using cationic materials such as dimethyldioctadecyl ammonium bromide (DDAB), cetyltrimethyl ammonium bromide(CTAB), We investigated the effect of cationic liposome formulations on in vitro DNA transfection, DDAB-containing liposomes showed increased transfection efficiency which was 3.2-fold as much as that by $Lipofectin^{\circledR}$, but CTAB-containing liposomes were inactive in gene transfection. The effect of colipid of DDAB-containing liposome was also investegated. As a colipid, dioleylphosphatidylethanolamine(DOPE) and cholesterol did altered the transfection efficiency of DDAB-containing liposomes. And increased DDAB concentration lowered the transfection efficiency. The optimum amount of liposomal formulation was $10\;{\mu}M$ for $1\;{\mu}g$ of DNA. In the experiment of stability, DOPE-containing liposomes formulation showed a broad size distribution and separation of two major peaks on a 5th day of preparation, but liposomes containing cholesterol was stable for 10 days. DDAB-containing liposomal DNA delivery system was prepared easily and was stable.

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STABLE TRANSFORMATION OF CULTURED CHICKEN CELLS

  • Han, J.Y.;Shin, Y.S.;Shoffner, R.N.;Guise, K.S.
    • Asian-Australasian Journal of Animal Sciences
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    • v.6 no.4
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    • pp.581-589
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    • 1993
  • A plasmid vector, $RSVLTR/{\beta}G2$, containing lacZ gene under the control of the RSVLTR promoter were transfected into chicken embryo fibroblasts by three different transfection methods. Calcium phosphate, lipsome and DEAE-dextran techniques were applied for transfection of chicken cells. A histochemical assay with X-gal was used as a simple method for screening transfected cells. Plasmid $RSVLTR/{\beta}G2$ was expressed proficiently in the chicken embryo fibroblast. Calcium phosphate-DNA precipitate transfection resulted in the highest efficiency for transient expression of $RSVLTR/{\beta}G2$. Transfected cells formed colonies on the 9th day of incubation indicating stable transformation of the inserted plasmid.

Comparative studies of various transfection processes for the optimal luminescence signal analysis (최적의 luminescence 신호 분석을 위한 유전자 전달 방법의 비교연구)

  • Park, Seohyun;Lee, Sunghou
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.17 no.11
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    • pp.640-647
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    • 2016
  • By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of $G{\alpha}16$ protein in order to track internal calcium mobilization by G protein-coupled receptors (GPCR). In this type of cell-based functional assay format, it is essential to optimize the transfection process of a receptor and $G{\alpha}16$ protein. For this study, corticotropin releasing factor receptor subtype 2(CRF2) was set as a model system to generate three stable cells with CRF2 and $G{\alpha}16$ in addition to transiently transfected cells under three different conditions. Agonist (sauvagine) and antagonist (K41498) responses in those cells were analyzed to develop the optimum transfection process. As a result, the effective signal ratio in the dose response experiments of sauvagine and K41498 were at least 10-fold higher (z'=0.77) in CRF2-$G{\alpha}16$ stable cells. For the transient transfection cells, stable expression of $G{\alpha}16$ prior to the CRF2 represented a two-fold higher signal (z'=0.84) than the other cases of transient transfection. In conclusion, for the utilization of transient transfection processes to develop a cell-based GPCR functional assay system, it is suggested to introduce various target receptors after stable expression of $G{\alpha}16$ protein.

Effect of Dexamethasone Preincubation on Polymer-Mediated Gene Delivery

  • Choi, Joon-Sig;Lee, Min-Hyung
    • Bulletin of the Korean Chemical Society
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    • v.26 no.8
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    • pp.1209-1213
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    • 2005
  • Nuclear membrane is one of the main barriers in intracellular delivery of genetic materials. The previous report showed that glucocorticoid receptor dilated the nuclear pore to 60 nm in the presence of a ligand. It was also suggested that the transport of genetic material to nucleus might be facilitated by glucocorticoid. In this study, the effect of glucocorticoid preincubation in the polymeric gene delivery was investigated. The cells were preincubated with dexamethasone, a potent glucocorticoid, and transfection assays were performed with polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer. As a result, the transfection efficiency of PEI or PAMAM to the cells in the presence of dexamethasone was enhanced, compared to the cells without dexamethasone. This effect was not observed in the cells preincubated with cholesterol. The polymer/DNA complex was stable in the presence of dexamethasone. In addition, the cytotoxicities of the polymeric carriers to the cells were observed in the presence of dexamethasone. In conclusion, dexamethasone enhances the transfection efficiency of polymeric carriers and may be useful in the development of polymeric gene carriers.

Sequential Conjugation of 6-Aminohexanoic Acids and L-Arginines to Poly(amidoamine) Dendrimer to Modify Hydrophobicity and Flexibility of the Polymeric Gene Carrier

  • Yu, Gwang-Sig;Yu, Ha-Na;Choe, Yun-Hui;Son, Sang-Jae;Ha, Tai-Hwan;Choi, Joon-Sig
    • Bulletin of the Korean Chemical Society
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    • v.32 no.2
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    • pp.651-655
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    • 2011
  • We synthesized a novel cationic dendrimer consisting of a poly(amidoamine) dendrimer (PAMAM, generation 4) backbone with both L-arginine (Arg) at the termini and 6-aminohexanoic acid (Ahx) between the original core polymer and the peripheral Arg units. The sequential chemical modification of PAMAM G4 with Ahx and Arg resulted in higher transfection efficiency with much less cytotoxicity. PAMAM G4-Ahx-Arg formed stable polyplexes at weight ratios of 8:1 or higher (polymer: plasmid DNA), and the mean polyplex diameter was $180{\pm}20nm$. PAMAM G4-Ahx-Arg showed much higher transfection ability than PAMAM G4 or PAMAM G4-Ahx. Furthermore, PAMAM G4-Ahx-Arg was much less cytotoxic than PEI25KD and PAMAM G4-Arg. In addition to Arg grafting of the PAMAM dendrimer, which endows a higher transfection capability, the addition of Ahx spacer increased dendrimer hydrophobicity, introduced flexibility into the conjugated amino acids, and reduced cytotoxicity. Overall, it appears that the concomitant modification of PAMAM with Ahx and Arg could lead to new PAMAM conjugates with better performances.

Transforming Capacity of the Plasmid Containing SV40 Promoter in NIH3T3 Fibroblast Cells (SV 40 Promoter를 갖는 Plasmid에 의한 NIH3T3 섬유아세포의 형질전환)

  • 이영환;김광식;서용택;김용웅;박남용;황태주
    • Korean Journal of Microbiology
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    • v.27 no.1
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    • pp.10-15
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    • 1989
  • The plasmid pKOneo, containing SV40 transcriptional promoter, has been used in the mouse tumorigenicity assay for oncogene studies. This assay employs a cotransfection of NIG3T3 fibroblast cells with the desired DNA and the plasmid pKOneo. This oncogene assay, however, has been speculated due to the SV40 transcriptional promoter in the plasmid pKOneo. This research was designed to investigate if the plasmid pKOneo alone is capable of transforming NiH3T3 fibroblast cells. The NIH3T3 subclones were established after the NIH3T3 cells were transfected with the plasmid pKOneo alone. The estabilished NIH3T3 subclones, containing the exogeneous plasmid pKOneo in their chromosomes, were examined for their expression of transformation-associated parameters. The results indicate that this plasmid pKOneo alone has positive effects on transformation of NIH3T3 cells after integration into cellular chromosomes.

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Establishment of An Efficient and Stable Transgene Expression System in Chicken Primordial Germ Cells

  • Yang, Ju-Hyun;Kim, Sung-Tae
    • Bulletin of the Korean Chemical Society
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    • v.33 no.5
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    • pp.1536-1540
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    • 2012
  • Chicken primordial germ cells (cPGCs) are founder germ cells in embryonic stage of development that eventually give rise to sperms or oocytes. Currently cPGCs are only known cells enabling germline transmission in chicken and their cultivation protocols were recently established. Although genome modifications of chickens are now theoretically possible using cPGCs, there are still several hurdles to overcome to practically use cPGCs as mediators for chicken transgenesis. First, efficiency of gene delivery into cPGCs remains low with current methods. Second, there aregene silencing mechanisms against the expression of foreign genes in cPGCs. In this study, we successfully increased the efficiency of gene delivery in cPGCs by taking advantage of the TTAA-specific $piggybac$ transposon system. Moreover, a pipette-type electroporator significantly enhanced transfection efficiency up to 5-fold compared withcuvette-type methods. Taken together, the technological advances in our study will provide practical benefits for the application to fulfill genetic modifications of chicken genome.

R3V6 Amphiphilic Peptide with High Mobility Group Box 1A Domain as an Efficient Carrier for Gene Delivery

  • Ryu, Jaehwan;Jeon, Pureum;Lee, Minhyung
    • Bulletin of the Korean Chemical Society
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    • v.34 no.12
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    • pp.3665-3670
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    • 2013
  • The R3V6 peptide includes a hydrophilic arginine stretch and a hydrophobic valine stretch. In previous studies, the R3V6 peptide was evaluated as a gene carrier and was found to have low cytotoxicity. However, the transfection efficiency of R3V6 was lower than that of poly-L-lysine (PLL) in N2A neuroblastoma cells. In this study, the transfection efficiency of R3V6 was improved in combination with high mobility group box 1A domain (HMGA). HMGA is originated from the nuclear protein and has many positively-charged amino acids. Therefore, HMGA binds to DNA via charge interaction. In addition, HMGA has a nuclear localization signal peptide and may increase the delivery efficiency of DNA into the nucleus. The ternary complex with HMGA, R3V6, and DNA was prepared and evaluated as a gene carrier. First, the HMGA/DNA complex was prepared with a negative surface charge. Then, R3V6 was added to the complex to coat the negative charges of the HMGA/DNA complex, forming the ternary complex of HMGA, R3V6, and DNA. A physical characterization study showed that the ternary complex was more stable than the PLL/DNA complex. The HMGA/R3V6/DNA complex had a higher transfection efficiency than the PLL/DNA, HMGA/DNA, or R3V6/DNA complexes in N2A cells. Furthermore, the HMGA/R3V6/DNA complex was not toxic to cells. Therefore, the HMGA/R3V6/DNA complex may be a useful gene delivery carrier.

Inflammatory Regulation by Haptoglobin in A549 Cells (A549 폐 상피세포에서 합토글로빈에 의한 염증반응 조절)

  • Kim Nam-Hoon;Lee Myung-Jae;Kim In-Sook
    • Journal of Life Science
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    • v.16 no.3
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    • pp.500-504
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    • 2006
  • Haptoglobin (Hp) is an acute phase protein, and its plasma level increases consistently in response to inflammation. To investigate the biological role of Hp in lung epithelial cells, the gene expressions of cyclooxygenase-2 (COX-2) and inflammatory cytokines were analyzed using human Hp gene-transfected A549 cells. Western blot analysis showed that COX-2 expression was markedly increased in Hp DNA-transfected cells (stable transfection and transient transfection) compared with that in vehicle DNA-transfected cells. When the Hp-expressing cells were treated with $1{\mu}g/ml$ of LPS or 100 U/ml of $IL-1{\beta}$ for 24 hr, the COX-2 expression was synergistically up-regulated. ACP-based PCR data demonstrated the Hp decreased SPARC expression, but increased IL-4 and S100AI expressions. These findings suggest that the Hp acts as a pro-inflammatory mediator in lung inflammation.

Establishment of an In Vitro TCD (Testosterone Compound Detection) System (테스토스테론 물질 검출을 위한 in vitro TCD 시스템 구축)

  • Lee, Dong-Geun;Jo, Jung-Kwon;Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.29 no.10
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    • pp.1159-1163
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    • 2019
  • Although there is a growing interest in male menopause, a phenomenon associated with male hormone depletion, current kits using antibodies to quantify male hormones are expensive. In this study, we constructed an in vitro system for verifying the activity or concentration of male steroid hormones using a transcriptional activity test. A reporter plasmid, pGL2-Neo-ARE-AdE1BTATA, which reacts to testosterone, was constructed. In this plasmid, the ARE-AdE1bTATA sequences can be bounded by the testosterone - androgen receptor complex to express luciferase as a reporter. Then, a stable transfection was performed on the human prostate cancer cell line, LNcap-LN3. The constructed LNcap-LN3/pGL2-Neo-ARE-AdE1BTATA testosterone compound detection (TCD) system showed quantitatively proportional luciferase activities to concentrations of $10^{-13}$ to $10^{-8}M$ of standard testosterone. The established in vitro TCD system will contribute to the development of materials for health/functional foods and drugs as it will be possible to search en masse for testosterone-like or testosterone-inhibiting substances derived from natural materials.