Evaluation and Optimization of Lipofectamine 3000 Reagents for Transient Gene Expression in KYSE-30 Esophagus Cancer Cell Line

Background and Aim: Transfection of DNA/RNA sequence into eukaryotic cells has a major effect on scientific studies. Various methods are used to transfer the DNA/RNA sequence into cells, such as lipid-based carriers as the available and easy procedure. Transfection with cationic lipid liposome is introduced as a simple and efficient procedure for monitoring the DNA/RNA sequence through gene function analysis, including fluorescence imaging RNA and protein expression. This study aimed to investigate the transfection efficiency and cell death through GFP expression in human esophageal squamous cell carcinoma (ESCC) cell line KYSE-30 using Lipofectamine 3000 reagent. Methods: The pCDH-513b plasmid DNA was transfected into KYSE-30 cells using Lipofectamine 3000 in different concentrations of the plasmid DNA and reagent. The transfection efficiency was evaluated by fluorescence microscope and flow cytometry analysis to determine the percentage of GFP-expressing cells. Moreover, the viability and death of transfected KYSE-30 cells were evaluated using a trypan blue exclusion assay. Results: The transfection efficiency of KYSE-30 with Lipofectamine 3000 was increased with higher plasmid DNA concentration and a lower amount of Lipofectamine 3000 reagent. The Optimized concentration of 1.5 µg plasmid DNA and volume of one µl of lipofectamine 3000 reagents were identified for 95% transfection efficiency in the KYSE-30 cell line. The viability and death of transfected cells were 43% and 58% after transfection, respectively. Conclusion: The results indicated that Lipofectamine 3000 might not be suitable for transfection in KYSE-30 cells due to increased cell death. *Corresponding Author: Mohammad Reza Abbaszadegan; Email: [email protected] Please cite this article as: Mahmoudian RA, Farshchian M, Abbaszadegan MR. Evaluation and Optimization of Lipofectamine 3000 Reagents for Transient Gene Expression in KYSE-30 Esophagus Cancer Cell Line. Arch Med Lab Sci. 2019;5(4):1-9. https://doi.org/10.22037/amls.v5i4.3108

Guanylyl Cyclase C as a tumor marker for detection of circulating tumor cells in the peripheral blood of colorectal cancer patients

Purpose: Guanylyl cyclase C (GCC) is one of the most frequent tumor markers to detect the circulating tumor cells (CTCs) in peripheral blood of colorectal cancer (CRC) patients. It has been proposed as a new marker for the molecular staging of CRC. The level of GCC mRNA expression in peripheral blood of CRC patients was evaluated to explore its probable correlations with the clinicopathological features.Methods: Relative quantitative expression analysis of GCC mRNA was performed on 80 blood samples (40 patients and 40 normal) using the Real-time RT-PCR.Results: GCC mRNA expression was detected in 70% of the CRC blood samples. The level of GCC mRNA expression in peripheral blood of patients was significantly higher than that in the normal cases (p = 0.031). Moreover, there was a significant correlation between the GCC copy number and advanced stages of tumor (p = 0.041). Furthermore, we have observed a significant correlation between tumor sizes and GCC copy numbers (p = 0.050).Conclusion: GCC can be a useful marker not only for detection of CTCs in CRC blood samples, but also for the molecular staging of colorectal cancer.

Cloning and Expression of Helicobacter pylori HpaA Gene

Objective: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, gastric adenocarcinomaand gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Antibiotictherapies do not protect from potential re-infection and have a risk for development of drugresistance. Therefore, prophylactic vaccine mediated protection against H. pylori is an attractiveclinical interest. H. pylori adhesin A (HpaA) is a conserved surface lipoprotein and playsimportant roles in the pathogenesis of infection. In this study the recombinant protein (rHpaA)was over-expressed in E.coli.Materials and Methods: The hpaA gene was amplified by PCR. Prokaryote expression vectorpET28a-hpaA was constructed, and used to transform E.coli BL21DE3. The expressionof recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot wereused to determine immunoreactivity of rHpaA by a rabbit polyclonal antibodies against wholecell of H. pylori.Results: The hpaA gene nucleotide sequence in the recombinant plasmid vector of pET-28-a-hpaA was consistent with that of H.pylori hpaA as published in the GenBank. SDS-PAGEdemonstrated that the constructed prokaryotic expression efficiently produced rHpaA at the1.5 mmol/L of IPTG. HpaA fusion protein was able to react with the rabbit polyclonal antibodyagainst whole cells of H. pylori.Conclusion: A prokaryotic expression system pET-28a-hpaA-BL21 with high efficiency of H.pylori hpaA gene was successfully established and the HpaA fusion protein showed satisfactoryimmunoreactivity. These results indicate that production of a specific recombinant proteinis an alternative and potentially more expeditious strategy for development of H. pylori vaccine

Role of Brg1 in progression of esophageal squamous cell carcinoma

Objective(s): Epigenetic regulation of gene expression can be carried out through chromatin remodeling enzymes such as SWI/SNF. Brg1 also known as SMARCA4 is a catalytic subunit of SWI/SNF, which is necessary for MMPs expression. Matrix metalloproteinases (MMPs) are known as important player enzymes during tumor progression and metastasis. Aberrant epigenetic modification of chromatin should be precisely clarified to reveal probable unknown pathways in ESCC progression. Probable role of Brg1 in ESCC tumorigenesis and metastasis was studied through the assessment of Brg1 mRNA expression in KYSE30, and further evaluation about the biology of Brg1 was performed through the Brg1 silencing. Materials and Methods: Level of Brg1 mRNA expression in KYSE30 was compared to normal tissues using the real time polymerase chain reaction (PCR). Moreover, KYSE30 cells were transfected with Brg1-siRNA to silence the Brg1. Results: Our results showed for the first time that Brg1 mRNA expression was increased in KYSE30 cell line (ESCC cell line) compared with normal esophageal tissue of ESCC patients. Rate of transfection in KYSE30 was also between 40 to 50%, using the pSilencer-Brg1shRNA (1:1 ratio). Conclusion: Our data indicated that chromatin remodeling machinery is a novel aspect in tumor biology of ESCC, and overexpression of Brg1 as an important member of SWI/SNF might be involved in the migration and invasion of ESCC tumoral cells

Genetic and Epigenetic landscape of Germline Stem Cells

Elucidating the critical epigenetics events involved in differentiation and reprogramming of cells to primordial germ cells (PGCs) is among the interesting issues in stem cell research. Here, I will talk about critical transcription factors and global hypomethylation in development of germ cells. Evidence strongly suggests that the earliest PGCs emerging in the E7.25 mouse embryo epiblast have a highly methylated genome, and high level of H3K9me2 in chromatin but during development, genome demethylated and patterns of histone codes changes dramatically. We designed a polycistroniclentiviral vector and overexpressed Stella, Oct4 and Nanos2 simultaneously in transduced cells; Increasing level of Prdm14, Nanog and decreasing of G9a expression is an interesting finding which might be considered as a primary step of reprogramming toward germline progenitor cells, here we propose decreasing H3K9me2 level as a consequence of G9a down regulation is a critical step which facilitated transition to different stemness state through creating a new epigenetic memory for the early germ cells.   Keywords: Epigenetic, hypomethylation, Germ line Stem Cells, polycistroniclentiviral vector

Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System

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