인간 대장암 세포 SW48과 RKO의 세포자멸사에 있어 의존수용체 RET9과 RET51 Transfection의 역할

서현일,박동일 대한장연구학회 2013 Intestinal Research Vol.11 No.1

목적: 의존수용체는 리간드의 결합유무에 따라서 세포의 증식, 분화, 이동 또는 세포자멸사를 촉진시키는 수용체이다. 이번 연구는 인간 대장암 세포주에서 의존수용체와 그 리간드를 이용하여 세포자멸사를 조절할 수 있는지 알아보기 위해 시행하였다. 방법: 세 가지 인간 대장암 세포주에 의존수용체 RET9과 RET51을 각각 transfection시킨 후 세포자멸사를 측정하였다. 동시에 의존수용체를 transfection시킨 12시간 후 리간드인 GDNF를 100 ng/mL 농도로 투여한 군의 세포자멸사도 측정하였다. Empty vector는 pcDNA를 사용하였다. 또한 GDNF 농도에 따른 세포자멸사도 측정하였다. 결과: RET51을 transfection시켰을 경우 SW48 세포주는 empty vector인 pcDNA를 transfection시킨 군에 비해 ELISA 검사 시 1.7배, caspase검사 시 2.19배 세포자멸사가 증가하였다. RKO 세포주는 ELISA 검사시 3.55배, caspase 검사 시 2.06배 증가하였다. V400 세포주는 세포자멸사의 증가가 관찰되지 않았다. RET9은 모든 세포주에서 세포자멸사의 증가가 관찰되지 않았다. RET51 transfection 12시간 후 GDNF를 투여한 SW48 세포주에서는 ELISA 검사 시 0.75배, caspase 검사 시 0.88배 측정되었으며, RKO 세포주에서는 각각 2.18배, 0.89배 측정되었다. RKO 세포주에 RET51을 transfection시킨 후 GDNF의 농도를 각각 0, 25, 50, 100 ng/mL로 투여하였을 때, 투여한 GDNF의 농도가높을수록 세포자멸사가 감소하는 것이 관찰되었다. 결론: 인간 대장암 세포주에 인위적으로 의존수용체와 그 리간드를 조작함으로서 세포자멸사를 촉진시킬 수도, 억제시킬 수도 있다는 것을증명함으로서 새로운 대장암 치료법을 시도해 볼 수 있는 이론적 근거를 마련하였다. Background/Aims: Dependent receptor can transmit both positive signal: proliferation, differentiation or migration; and negative signal: apoptosis. It depends on the presence of its ligand. This study was performed to determine the effects of transfection of dependent receptors in human colon cancer cell lines. Methods: Two dependent receptors (rearranged during transfection [RET]9 and RET51) were transfected into three human colon cancer cell lines: SW48, RKO and V400. Then, half of them were treated with glial cell line-derived neurotrophic factor (GDNF). Using ELISA and caspase assay, apoptosis was measured. Dose-response relation between GDNF and apoptosis was also analyzed. A pcDNA was used as an empty vector. Results:After transfection of RET51, apoptosis was increased in SW48 (70% with ELISA and 119% with caspase assay) and RKO (255% with ELISA and 106% with caspase assay) cell lines when compared with the pcDNA group. V400 cell line did not show increased apoptosis. Transfection of RET9 did not induce apoptosis in all of the three human colon cancer cell lines. Treatment with GDNF 12 hours after transfection of RET51 decreased apoptosis in SW48 (66% with ELISA and 60% with caspase assay) and RKO (39% with ELISA and 57% with caspase assay) when compared with the cell lines transfected with RET51 only. Apoptosis was down-regulated with increasing concentration of GDNF in RKO cell line. Conclusions: This study showed that the apoptosis of human colon cancer cell line can be controlled by manipulating the dependent receptors and its ligands. We present the possibility of therapeutic method using dependent receptor in colon cancer. (Intest Res 2013;11:28-33)

Optomizing Transfection Efficiency of Cervical Cancer Cells Transfected by Cationic Liposomes Lipofectamine^TM2000

Huang, Fei,Zhao, Feng,Liang, Li-Ping,Zhou, Mei,Qu, Zhi-Ling,Cao, Yan-Zhen,Lin, Chen Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.17

Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Materials and Methods: $Lipofectamine^{TM}2000$ as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA, miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. Results: The seeding density of Hela cell line and Siha are $1.5{\times}10^4$ (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01). MTT assay showed that according to the above conditions which has the lowest cytotoxicity. Conclusions: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection

Lee Myoung Hui,Lee Jiyoung,Choi Seung A,Kim Ye-Sol,Koo Okjae,Choi Seung Hee,Ahn Woo Seok,Jie Eun Yee,Kim Suk Weon 한국식물생명공학회 2020 Plant biotechnology reports Vol.14 No.6

Nowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR–Cas9) nuclease system. However, to combine protoplast technology with the CRISPR–Cas9 system in plants, a stable and an efcient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR–Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7±2.5% and 52±7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3–Cas9 protein delivery into the nucleus was confrmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efcient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.

Improved Transfection Efficiency and Metabolic Activity in Human Embryonic Stem Cell Using Non-Enzymatic Method

Kim, C-Yoon,Hwang, In-Kyu,Kang, Changhee,Chung, Eun-Bin,Jung, Cho-Rok,Oh, Hanseul,Jeong, Young-Hoon,Moon, Sung-Hwan,Kim, Jong Soo,Hong, Ki-Sung,Park, Jae-Hak,Chung, Hyung-Min Korean Society for Stem Cell Research 2018 International journal of stem cells Vol.11 No.2

<P>Human embryonic stem cells (hESCs) are pluripotent cells widely used in conventional and regenerative medicine due to their ability to self-renew, proliferate and differentiate. Recently, genetic modification of stem cells using genome editing is the most advanced technique for treating hereditary diseases. Nevertheless, the low transfection efficiency of hESCs using enzymatic methods is still limited in <I>in vitro</I> preclinical research. To overcome these limitations, we have developed transfection methods using non-enzymatic treatments on hESCs. In this study, hESCs were transfected following enzymatic (TrypLE and trypsin) and non-enzymatic treatment ethylenediaminetetraacetic acid (EDTA) to increase transfection efficiency. Flow cytometric analysis using an enhanced green fluorescent protein vector showed a significantly increased transfection efficiency of EDTA method compared to standard enzyme method. In addition, the EDTA approach maintained stable cell viability and recovery rate of hESCs after transfection. Also, metabolic activity by using Extracellular Flux Analyzer revealed that EDTA method maintained as similar levels of cell functionality as normal group comparing with enzymatic groups. These results suggest that transfection using EDTA is a more efficient and safe substitute for transfection than the use of standard enzymatic methods.</P>

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

Lee, Sung-Lim,Ock, Sun-A,Yoo, Jae-Gyu,Kumar, B. Mohana,Choe, Sang-Yong,Rho, Gyu-Jin Wiley Subscription Services, Inc., A Wiley Company 2005 Molecular reproduction and development Vol.72 No.2

<P>The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene<SUP>®</SUP> (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%–5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5–7 and 20–22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 ± 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 ± 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 ± 1.7 and 5.1 ± 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 ± 1.5 and 3.3 ± 0.8, 8.8 ± 0.7, and 2.1 ± 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. ∼110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.</P>

Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

서민구,손성림,김지원,윤건호,이숙경 대한당뇨병학회 2011 Diabetes and Metabolism Journal Vol.35 No.1

Background: Previously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF)gene in an Epstein-Barr virus (EBV)-based plasmid (pEBVHGF) showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model. Methods: Neonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry. Results: Re-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF). Conclusion: For clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

대장상피세포에서 Interleukin-10 유전자 전달의 CXC 케모카인에 대한 억제 효과

이국래 ( Kook Lae Lee ),김찬규 ( Chan Gyoo Kim ),김병관 ( Byeong Gwan Kim ),장동경 ( Dong Kyung Chang ),이동호 ( Dong Ho Lee ),김주성 ( Joo Sung Kim ),정현채 ( Hyun Chae Jung ),이연 ( Yeon Lee ),박종상 ( Jong Sang Park ),송인성 ( 대한소화기학회 2003 대한소화기학회지 Vol.41 No.6

Background/Aims: Cytokine plays an important role in initiation and continuation of inflammatory bowel disease. However, cytokine protein has some limitation as a therapeutic tool because of low bioavailability, poor pharmacokinetics and chemical instability. Thus, we studied the effect of interleukin 10 (IL-10) gene transfection on murine colon cancer cell line by using non-viral gene carrier. Methods: Therapeutic gene and plasmid was pCAGGS mouse IL-10 and gene carriers were polyethyleneimine (PEI) and 3β[L-ornithinamide-carbamoyl] cholesterol (O-chol). After IL-10 gene transfection, we measured the level of IL-10 in supernatant of cultured CT-26 cells. The chemokine cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein (MIP)-2, which were treated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), were measured after IL-10 gene transfection. Results: The IL-10 values were increased significantly by using PEI, but not by using O-chol. The KC and MIP-2 values were increased when LPS or TNF-α were treated. When PEI was used, KC and MIP-2 values increased by LPS or TNF-α were decreased. When O-chol was used, the KC values increased by TNF-α were decreased but those treated by LPS were not decreased, and the MIP-2 values were not decreased. Conclusions: After IL-10 gene transfection in colon cancer cell, IL-10 cytokine was efficiently expressed. The increased chemokine values by LPS or TNF-α were suppressed by IL-10 gene transfection, but which was not constant because of carrier efficiency. (Korean J Gastroenterol 2003;41:447-455)

Preparation and Gene Delivery of Alkaline Amino Acids-Based Cationic Liposomes

Li, Li,Nie, Yu,Zhu, Rong,Shi, Sanjun,Luo, Kui,He, Bin,Yang, Yang,Yang, Junxiao,Gu, Zhongwei 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.7

Cationic lipids 1, 2, and 3, based on hydrophobic cholesterol linked to L-lysine, L-histidine or Larginine, respectively, were designed and tested as gene delivery vectors. Physicochemical and biological properties of all liposomes and lipoplexes were evaluated, including lipid-DNA interactions, size, morphology, zeta potential, acid-base buffering capability, protection of DNA from DNase I digestion, and cytotoxity. The efficiency of luciferase gene transfection of lipoplexes 1-3 was compared with that of commercial dioleoyl-trimethylammonium propane (DOTAP) and polyethyleneimine (PEI) in 293T cells and HepG2 cells with or without poly(ethylene glycol) PEG stabilizer. The complexation and protection of DNA of liposome 3 was the strongest among the three liposomes. The efficiency of gene transfection of liposomes 1-3 was two-to threefold higher than that of PEI and/or DOTAP in 293T cells. Liposomes 1 and 3 in PEG as stabilizer showed sixfold higher transfection efficiency than that of PEI and/or DOTAP, whereas liposome 2 showed very low transfection efficiency. In HepG2 cells, the transfection efficiency of all the cationic liposomes was much lower than that of DOTAP. In conclusion, lipids 1-3 were efficient and non-toxic gene vectors; the headgroup of cationic lipids and the stabilizer of liposome formulation had an important influence on gene transfection.

최적의 luminescence 신호 분석을 위한 유전자 전달 방법의 비교연구

박서현(Seohyun Park),이승호(Sunghou Lee) 한국산학기술학회 2016 한국산학기술학회논문지 Vol.17 No.11

형광 간섭 현상을 최소화시켜 상대적으로 민감한 측정이 가능한 aequorin기반 luminescence기술은 Gα16 단백질 도입을 통해 세포 내부의 칼슘 이동 신호를 감지하여 G 단백질 결합 수용체(G protein-coupled receptor, GPCR)의 기능 분석을 가능하게 하는 세포 기반 분석 기술로 수용체 및 G 단백질 유전자 전달의 최적화 과정이 필수적이다. 본 연구를 위해 corticotropin releasing factor receptor subtype 2(CRF2) 수용체를 모델 시스템으로 CRF2와 Gα16 단백질이 구축된 세 가지 안정화 세포주를 제작하였고, 이들을 이용한 서로 다른 세 가지 조건의 임시 발현 세포주에서 작용제(sauvagine)와 길항제(K41498)의 반응성을 분석하여 최적의 유전자 전달 방법을 도출하고자 하였다. 그 결과 sauvagine 및 K41498의 농도에 따른 반응에서 CRF2-Gα16 안정화 세포주가 임시 발현 세포주보다 10배 이상의 유효신호 비율을 나타내었고(z’=0.77) 임시 발현 세포주의 경우 Gα16의 안정화 발현 이후에 CRF2를 전달하는 경우가 다른 임시 발현 조건보다 2배 이상 높은 효율을 보였다(z’=0.84). 따라서 임시 유전자 전달 기술을 GPCR 세포 기능 분석 시스템에 활용할 경우 Gα16 단백질에 대한 안정화 세포주를 우선적으로 구축하고, 목표하는 다양한 수용체들을 단계적으로 발현시키는 것이 최선의 방법이라 판단된다. By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of Gα16 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 Gα16 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 Gα16 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-Gα16 stable cells. For the transient transfection cells, stable expression of Gα16 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 Gα16 protein.

Enhanced transfection by antioxidative polymeric micelles that reduces polyplex-mediated cellular oxidative Stress

이민상,김낙원,정지훈 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

Recently, the potential toxicity of nanomaterials has attracted considerable attention since materials in nano-scale dimensions often display substantially different properties compared to bulk materials with the same composition and may have unexpected adverse effects on tissues and cells. Various inorganic or organic nanoparticles with diameters of a few hundred nanometers have been reported to elicit inflammatory responses, oxidative injury, cellular cytotoxicity, or genotoxicity. A major pathway responsible for these adverse effects is thought to involve oxidative stress, which is often the result of excess intracellular reactive oxygen species (ROS). In this study, we designed an antioxidative transfection system minimizing cellular oxidative stress for enhanced transfection.