• Genomics & Informatics
• /
• 제9권4호
• /
• pp.161-172
• /
• 2011

Angiogenesis is regulated by a large number of molecules and complex signaling mechanisms. The G protein $G{\alpha}_{13}$ is a part of this signaling mechanism as an endothelial cell movement regulator. Gene expression analysis of $G{\alpha}_{13}$ knockout mouse embryos was carried out to identify the role of $G{\alpha}_{13}$ in angiogenesis signaling during embryonic development. Hypoxia-inducible response factors including those acting as regulators of angiogenesis were over expressed, while genes related to the cell cycle, DNA replication, protein modification and cell-cell dissociation were under expressed. Functional annotation and network analysis indicate that $G{\alpha}_{13}{^{-/-}}$ embryonic mice were exposed to hypoxic conditions. The present analysis of the time course highlighted the significantly high levels of disorder in the development of the cardiovascular system. The data suggested that hypoxia-inducible factors including those associated with angiogenesis and abnormalities related to endothelial cell division contributed to the developmental failure of $G{\alpha}_{13}$ knockout mouse embryos.

• Bulletin of the Korean Chemical Society
• /
• 제28권8호
• /
• pp.1317-1321
• /
• 2007

Arginine conjugated PAMAM dendrimer, PAMAM-R was modified with propylene oxide via hydroxylation of primary amines of arginine residues. About 49 amines were detected to be converted to amino alcohols by 1H NMR. The newly synthesized polymer, PAMAM-R-PO was able to completely retard pDNA from a charge ratio of 2. The average diameter of PAMAM-R-PO polyplex was found to be 242 nm at a charge ratio of 30. The Zeta-potential value of PAMAM-R-PO polyplex was able to reach 20-30 mV over a charge ratio of 10. PAMAM-R-PO indicated higher cell viability than unmodified PAMAM-R on HeLa and 293 cells because of its hydroxylated amines. Transfection experiments on 293 cells showed that the transfection efficiency of PAMAM-R-PO was found to be 1.5-1.9 times higher than that of PEI25kDa at a charge ratio of 30. The polymer eventually displayed about 2 times greater transfection efficiency than PAMAM-R at the same charge ratio in the absence of serum. Therefore, we concluded that the modification of primary amines of PAMAMR to amino alcohols gives positive effects such as reduced cytotoxicity and enhanced transfection efficiency on 293 cells for gene delivery potency of PAMAM-R.

• Journal of Pharmaceutical Investigation
• /
• 제40권6호
• /
• pp.333-337
• /
• 2010

Combining cell- and gene-based therapy is a promising therapeutic strategy in regenerative medicine. The aim of this study was to develop genetically modified mesenchymal stem cell (MSC) aggregates using a poly(ethylene glycol) (PEG) hydrogel micro-well array technique. Stable PEG hydrogel micro-well arrays with diameters of 200 to $500\;{\mu}m$ were fabricated and used to generate genetically engineered MSC aggregates. Rat bone marrow-derived MSCs were transfected with a green fluorescent protein (GFP) plasmid as a reporter gene, and aggregated by culturing in the PEG hydrogel micro-well arrays. The resultant cell aggregates had a mean diameter of less than $200\;{\mu}m$, and maintained the mesenchymal phenotype even after genetic modification and cell aggregation. Transplantation of MSC aggregates that are genetically modified to express therapeutic or cell-survival genes may be a potential therapeutic approach for regenerative medicine.

• Genes and Genomics
• /
• 제40권11호
• /
• pp.1119-1125
• /
• 2018

Melanocortin 4 receptor: (MC4R) and Myostatin (MSTN) are two important growth trait-related genes in animals. In this study, we showed that two SNPs, MC4R-719A>G and MSTN-519C>T, found in the promoters of the MC4R and MSTN genes, respectively, are both associated with growth traits in Spinibarbus hollandi. Furthermore, we observed that there were significant associations between the expression levels of the MC4R and MSTN genes and these two growth trait-related SNPs. The expression level of MC4R gene in brain was lower in GG genotype fish with extremely high growth performance than that in AA genotype fish with extremely low growth performance. Expression level of the MSTN gene in muscle was lower in TT genotype fish with extremely high growth performance than that in CC and CT genotype fish with lower growth performance. The results indicated that these SNPs located in the promoters of MC4R and MSTN are associated with growth-related traits through modification of gene expression levels. The MSTN and MC4R SNPs may have useful application in effective marker-assisted selection aimed to increase output in S. hollandi.

• Asian-Australasian Journal of Animal Sciences
• /
• 제33권6호
• /
• pp.1023-1033
• /
• 2020

Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

• Reproductive and Developmental Biology
• /
• 제33권1호
• /
• pp.63-69
• /
• 2009

In this study we tried to construct a more efficient tetracycline-inducible gene expression system by replacing previous key elements with more advance ones. At the beginning, we substituted PGK (phophoglycerate kinase) promoter for CMV (cytomegalovirus) promoter to control "$rtTA2^sM2$" which has been known for high induction efficiency in response to tetracycline. With this modification, expression of the EGFP marker gene under the induction condition was significantly increased. Next, we replaced "TRE" fragment with a modified version named "TRE-tighf" which has been reported to have higher affinity and specificity to the transactivator by minor base change of the "TRE" DNA fragment sequence. Use of "TRE-tighf" instead of "TRE" resulted in more than 10 fold increment in terms of induction efficiency and significant decrement of background expression in non-inducible condition. By combining PGK promoter and "TRE-tight" fragment, we could upgrade previous tetracycline-inducible system to show more stringent turn on/off gene switch ability and stronger expression of the gene of our interest. Use of this newly developed system must be very helpful to the studies of gene expression, especially to the transgenic animal study in which non-controllable constitutive expression of the transgene has been one of the urgent problems to be solved.

• Journal of Microbiology and Biotechnology
• /
• 제20권5호
• /
• pp.917-924
• /
• 2010

The practicability of transgenic tobacco engineered to express bacterial native mercuric reductase (MerA), responsible for the transport of $Hg^{2+}$ ions into the cell and their reduction to elemental mercury ($Hg^0$), without any codon modification, for phytoremediation of mercury pollution was evaluated. Transgenic tobacco plants reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve the less toxic elemental mercury. Transformed tobacco produced a large amount of merA protein in leaves and showed a relatively higher resistance phenotype to $HgCl_2$ than wild type. Results suggest that the integrated merA gene, encoding mercuric reductase, a key enzyme of the bacterial mer operon, was stably integrated into the tobacco genome and translated to active MerA, which catalyzes the bioconversion of toxic $Hg^{2+}$ to the least toxic elemental $Hg^0$, and suggest that MerA is capable of reducing the $Hg^{2+}$, probably via NADPH as an electron donor. The transgenic tobacco expressing merA volatilized significantly more mercury than wild-type plants. This is first time we are reporting the expression of a bacterial native merA gene via the nuclear genome of Nicotiana tabacum, and enhanced mercury volatilization from tobacco transgenics. The study clearly indicates that transgenic tobacco plants are reasonable candidates for the remediation of mercurycontaminated areas.

• Genomics & Informatics
• /
• 제19권2호
• /
• pp.17.1-17.13
• /
• 2021

Breast cancer is one of the leading causes of cancer in women all over the world and accounts for ~25% of newly observed cancers in women. Epigenetic modifications influence differential expression of genes through non-coding RNA and play a crucial role in cancer regulation. In the present study, epigenetic regulation of gene expression by in-silico analysis of histone modifications using chromatin immunoprecipitation sequencing (ChIP-Seq) has been carried out. Histone modification data of H3K4me3 from one normal-like and four breast cancer cell lines were used to predict miRNA expression at the promoter level. Predicted miRNA promoters (based on ChIP-Seq) were used as a probe to identify gene targets. Five triple-negative breast cancer (TNBC)-specific miRNAs (miR153-1, miR4767, miR4487, miR6720, and miR-LET7I) were identified and corresponding 13 gene targets were predicted. Eight miRNA promoter peaks were predicted to be differentially expressed in at least three breast cancer cell lines (miR4512, miR6791, miR330, miR3180-3, miR6080, miR5787, miR6733, and miR3613). A total of 44 gene targets were identified based on the 3'-untranslated regions of downregulated mRNA genes that contain putative binding targets to these eight miRNAs. These include 17 and 15 genes in luminal-A type and TNBC respectively, that have been reported to be associated with breast cancer regulation. Of the remaining 12 genes, seven (A4GALT, C2ORF74, HRCT1, ZC4H2, ZNF512, ZNF655, and ZNF608) show similar relative expression profiles in large patient samples and other breast cancer cell lines thereby giving insight into predicted role of H3K4me3 mediated gene regulation via the miRNA-mRNA axis.

• Journal of Microbiology
• /
• 제35권4호
• /
• pp.300-308
• /
• 1997

The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.

• Bulletin of the Korean Chemical Society
• /
• 제32권2호
• /
• pp.651-655
• /
• 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.