Displaying publications 21 - 40 of 79 in total

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  1. Fulltext NFIX as a Master Regulator for Lung Cancer Progression
    Rahman NIA, Abdul Murad NA, Mollah MM, Jamal R, Harun R
    Front Pharmacol, 2017;8:540.
    PMID: 28871224 DOI: 10.3389/fphar.2017.00540
    About 40% of lung cancer cases globally are diagnosed at the advanced stage. Lung cancer has a high mortality and overall survival in stage I disease is only 70%. This study was aimed at finding a candidate of transcription regulator that initiates the mechanism for metastasis by integrating computational and functional studies. The genes involved in lung cancer were retrieved using in silico software. 10 kb promoter sequences upstream were scanned for the master regulator. Transient transfection of shRNA NFIXs were conducted against A549 and NCI-H1299 cell lines. qRT-PCR and functional assays for cell proliferation, migration and invasion were carried out to validate the involvement of NFIX in metastasis. Genome-wide gene expression microarray using a HumanHT-12v4.0 Expression BeadChip Kit was performed to identify differentially expressed genes and construct a new regulatory network. The in silico analysis identified NFIX as a master regulator and is strongly associated with 17 genes involved in the migration and invasion pathways including IL6ST, TIMP1 and ITGB1. Silencing of NFIX showed reduced expression of IL6ST, TIMP1 and ITGB1 as well as the cellular proliferation, migration and invasion processes. The data was integrated with the in silico analyses to find the differentially expressed genes. Microarray analysis showed that 18 genes were expressed differentially in both cell lines after statistical analyses integration between t-test, LIMMA and ANOVA with Benjamini-Hochberg adjustment at p-value < 0.05. A transcriptional regulatory network was created using all 18 genes, the existing regulated genes including the new genes PTCH1, NFAT5 and GGCX that were found highly associated with NFIX, the master regulator of metastasis. This study suggests that NFIX is a promising target for therapeutic intervention that is expected to inhibit metastatic recurrence and improve survival rate.
    Matched MeSH terms: Transfection
  2. Development of a T7 RNA polymerase expressing cell line using lentivirus vectors for the recovery of recombinant Newcastle disease virus
    Yeong MY, Cheow PS, Abdullah S, Song AA, Lei-Rossmann J, Tan TK, et al.
    J Virol Methods, 2021 05;291:114099.
    PMID: 33592218 DOI: 10.1016/j.jviromet.2021.114099
    The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.
    Matched MeSH terms: Transfection
  3. In vitro expression of erythropoietin by transfected human mesenchymal stromal cells
    Mok PL, Cheong SK, Leong CF, Othman A
    Cytotherapy, 2008;10(2):116-24.
    PMID: 18368590 DOI: 10.1080/14653240701816996
    Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro.
    Matched MeSH terms: Transfection*
  4. Matrix Production in Chondrocytes Transfected with Sex Determining Region Y-Box 9 and Telomerase Reverse Transcriptase Genes: An In Vitro Evaluation from Monolayer Culture to Three-Dimensional Culture
    Md Nazir N, Zulkifly AH, Khalid KA, Zainol I, Zamli Z, Sha'ban M
    Tissue Eng Regen Med, 2019 06;16(3):285-299.
    PMID: 31205857 DOI: 10.1007/s13770-019-00191-1
    Background: This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture.

    Methods: The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D "cells-scaffolds" constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction.

    Results: All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D "cells-scaffolds" constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA.

    Conclusion: These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.

    Matched MeSH terms: Transfection*
  5. Fulltext Evaluation of two transformation protocols and screening of positive plasmid introduction into Bacillus cereus EB2, a gram-positive bacterium using qualitative analyses
    Sirajuddin SA, Sundram S
    Braz J Microbiol, 2020 Sep;51(3):919-929.
    PMID: 32078730 DOI: 10.1007/s42770-020-00241-0
    Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.
    Matched MeSH terms: Transfection/methods*
  6. Fulltext Advancements in reprogramming strategies for the generation of induced pluripotent stem cells
    Lai MI, Wendy-Yeo WY, Ramasamy R, Nordin N, Rosli R, Veerakumarasivam A, et al.
    J Assist Reprod Genet, 2011 Apr;28(4):291-301.
    PMID: 21384252 DOI: 10.1007/s10815-011-9552-6
    Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has emerged as an invaluable method for generating patient-specific stem cells of any lineage without the use of embryonic materials. Following the first reported generation of iPS cells from murine fibroblasts using retroviral transduction of a defined set of transcription factors, various new strategies have been developed to improve and refine the reprogramming technology. Recent developments provide optimism that the generation of safe iPS cells without any genomic modification could be derived in the near future for the use in clinical settings. This review summarizes current and evolving strategies in the generation of iPS cells, including types of somatic cells for reprogramming, variations of reprogramming genes, reprogramming methods, and how the advancement iPS cells technology can lead to the future success of reproductive medicine.
    Matched MeSH terms: Transfection/methods*
  7. Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency
    Akinsola RO, Lee CW, Sim EUH, Narayanan K
    Anal Biochem, 2021 03 01;616:114088.
    PMID: 33358938 DOI: 10.1016/j.ab.2020.114088
    Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.
    Matched MeSH terms: Transfection/methods
  8. Roles of MicroRNA21 and MicroRNA29a in Regulating Cell Adhesion Related Genes in Bone Metastasis Secondary to Prostate Cancer
    Mohamad M, Wahab NA, Yunus R, Murad NA, Zainuddin ZM, Sundaram M, et al.
    Asian Pac J Cancer Prev, 2016;17(7):3437-45.
    PMID: 27509989
    BACKGROUND: There is an increasing concern in the role of microRNA (miRNA) in the pathogenesis of bone metastasis (BM) secondary to prostate cancer (CaP). In this exploratory study, we hypothesized that the expression of vinculin (VCL) and chemokine X3C ligand 1 (CX3CL1) might be downregulated in clinical samples, most likely due to the posttranscriptional modification by microRNAs. Targeted genes would be upregulated upon transfection of the bone metastatic prostate cancer cell line, PC3, with specific microRNA inhibitors.

    MATERIALS AND METHODS: MicroRNA software predicted that miR21 targets VCL while miR29a targets CX3CL1. Twenty benign prostatic hyperplasia (BPH) and 16 high grade CaP formalinfixed paraffin embedded (FFPE) specimens were analysed. From the bone scan results, high grade CaP samples were further classified into CaP with no BM and CaP with BM. Transient transfection with respective microRNA inhibitors was done in both RWPE1 (normal) and PC3 cell lines. QPCR was performed in all FFPE samples and transfected cell lines to measure VCL and CX3CL1 levels.

    RESULTS: QPCR confirmed that VCL messenger RNA (mRNA) was significantly down regulated while CX3CL1 was upregulated in all FFPE specimens. Transient transfection with microRNA inhibitors in PC3 cells followed by qPCR of the targeted genes showed that VCL mRNA was significantly up regulated while CX3CL1 mRNA was significantly downregulated compared to the RWPE1 case.

    CONCLUSIONS: The downregulation of VCL in FFPE specimens is most likely regulated by miR21 based on the in vitro evidence but the exact mechanism of how miR21 can regulate VCL is unclear. Upregulated in CaP, CX3CL1 was found not regulated by miR29a. More microRNA screening is required to understand the regulation of this chemokine in CaP with bone metastasis. Understanding miRNAmRNA interactions may provide additional knowledge for individualized study of cancers.

    Matched MeSH terms: Transfection/methods
  9. Nucleotide sequencing, cloning, and expression of Capra hircus Heme Oxygenase-1 in caprine islets to promote insulin secretion in vitro
    Vakhshiteh F, Allaudin ZN, Lila MA, Abbasiliasi S, Ajdari Z
    Mol Biotechnol, 2015 Jan;57(1):75-83.
    PMID: 25218408 DOI: 10.1007/s12033-014-9803-8
    Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.
    Matched MeSH terms: Transfection
  10. Silencing of myeloid cell leukemia-1 by small interfering RNA improves chemosensitivity to etoposide in u-937 leukemic cells
    Jafarlou M, Baradaran B, Shanehbandi D, Saedi TA, Jafarlou V, Karimi P, et al.
    J Biol Regul Homeost Agents, 2016 Jan-Mar;30(1):55-65.
    PMID: 27049076
    A key issue in the treatment of acute myeloid leukemia (AML) is the development of drug resistance to chemotherapeutic agents. Overexpression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein, is associated with tumor progression and drug resistance in leukemia and several cancers. The purpose of this study was to investigate the effect of specific Mcl-1 small interference RNA (siRNA) on the proliferation and chemosensitivity of U-937 AML cell to etoposide. The siRNA transfection was conducted using Lipofectamine™ 2000. Quantitative real-time RT-PCR (qRT-PCR) and Western blot analysis were employed to measure the expression levels of mRNA and protein, respectively. To evaluate tumor cell growth after siRNA transfection, Trypan blue exclusion assay was conducted. The cytotoxic effects of siRNA and etoposide were determined using MTT assay on their own and in combination. DNA-histone ELISA and annexin-V/FITC assays were performed to study the apoptosis. Mcl-1 siRNA transfection significantly blocked the expression of Mcl-1 mRNA and protein in a time-dependent manner, leading to a strong growth inhibition and enhanced apoptosis (P less than 0.05). Furthermore, pretreatment with Mcl-1 siRNA, synergistically enhanced the cytotoxic and apoptotic effects of etoposide (P less than 0.05). Our results demonstrated that Mcl-1 plays a fundamental role in the survival and resistance of U-937 cells to etoposide. Therefore, Mcl-1 can be considered an attractive target in gene therapy of AML patients and siRNA-mediated silencing of this gene may be a novel strategy in AML treatment.
    Matched MeSH terms: Transfection
  11. Dysregulation of non-histone molecule miR205 and LRG1 post-transcriptional de-regulation by SETD1A in triple negative breast cancer
    Mohamad Hanif EA
    Mol Biol Rep, 2019 Dec;46(6):6617-6624.
    PMID: 31552595 DOI: 10.1007/s11033-019-05079-w
    FEC chemo-resistance in triple negative breast cancer (TNBC) remains a challenge. Therefore it is crucial to determine the right treatment regime by understanding molecular mechanisms of driver regulators involved in the progression of TNBCs. This study aims to understand SETD1A mechanisms in TNBC development in two TNBC cell lines. SETD1A was transiently transfected in MDA-MB-468 (FEC good prognosis) and Hs578T (FEC poor prognosis). Regulation of potential targets miR205, EMT marker ZEB1 and LRG1 and proliferative marker Ki-67 were tested by RqPCR to elucidate SETD1A interactions. This study displayed significant recovery of miR205 with SETD1A depletion and reduction of ZEB1 in MDA-MB-468. However, ZEB1 remained unchanged in Hs578T indicating ZEB1 regulation may be outcompeted by other mechanisms associated with aggressive cell line characteristics and the expression of endogenous ZEB1 was relatively high in Hs578T. Elevation of LRG1 and declined Ki-67 were observed by SETD1A knocked down. Enhanced expression was observed by LRG1 in Hs578T and not in MDA-MB-468 suggesting LRG1 contributed to distinct poor FEC outcome in TNBCs. The underlying mechanism of SETD1A in miR205/ZEB1/Ki-67/LRG1 axis needs further evaluation. Whether abrogation of the pathway is indeed associated with transcriptional or post-transcriptional activation in TNBC cell lines models, clearly validation in clinical samples is warranted to achieve its prognostic and therapeutic values in TNBCs.
    Matched MeSH terms: Transfection
  12. Fulltext Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment
    Habib O, Mohd Sakri R, Ghazalli N, Chau DM, Ling KH, Abdullah S
    PLoS One, 2020;15(12):e0244386.
    PMID: 33347482 DOI: 10.1371/journal.pone.0244386
    CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.
    Matched MeSH terms: Transfection
  13. Fulltext MicroRNA-124a inhibits endoderm lineage commitment by targeting Sox17 and Gata6 in mouse embryonic stem cells
    Liew LC, Gailhouste L, Tan GC, Yamamoto Y, Takeshita F, Nakagama H, et al.
    Stem Cells, 2020 04;38(4):504-515.
    PMID: 31828873 DOI: 10.1002/stem.3136
    The role of microRNAs (miRNAs) during mouse early development, especially in endoderm germ layer formation, is largely unknown. Here, via miRNA profiling during endoderm differentiation, we discovered that miR-124a negatively regulates endoderm lineage commitment in mouse embryonic stem cells (mESCs). To further investigate the functional role of miR-124a in early stages of differentiation, transfection of embryoid bodies with miR-124a mimic was performed. We showed that overexpression of miR-124a inhibits endoderm differentiation in vitro through targeting the 3'-untranslated region (UTR) of Sox17 and Gata6, revealing the existence of interplay between miR-124a and the Sox17/Gata6 transcription factors in hepato-specific gene regulation. In addition, we presented a feasible in vivo system that utilizes teratoma and gene expression profiling from microarray to quantitatively evaluate the functional role of miRNA in lineage specification. We demonstrated that ectopic expression of miR-124a in teratomas by intratumor delivery of miR-124a mimic and Atelocollagen, significantly suppressed endoderm and mesoderm lineage differentiation while augmenting the differentiation into ectoderm lineage. Collectively, our findings suggest that miR-124a plays a significant role in mESCs lineage commitment.
    Matched MeSH terms: Transfection
  14. Fulltext Electrosprayed Alginate Nanoparticles as CRISPR Plasmid DNA Delivery Carrier: Preparation, Optimization, and Characterization
    Alallam B, Altahhan S, Taher M, Mohd Nasir MH, Doolaanea AA
    Pharmaceuticals (Basel), 2020 Jul 22;13(8).
    PMID: 32707857 DOI: 10.3390/ph13080158
    Therapeutic gene editing is becoming more feasible with the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system. However, the successful implementation of CRISPR/Cas9-based therapeutics requires a safe and efficient in vivo delivery of the CRISPR components, which remains challenging. This study presents successful preparation, optimization, and characterization of alginate nanoparticles (ALG NPs), loaded with two CRISPR plasmids, using electrospray technique. The aim of this delivery system is to edit a target gene in another plasmid (green fluorescent protein (GFP)). The effect of formulation and process variables were evaluated. CRISPR ALG NPs showed mean size and zeta potential of 228 nm and -4.42 mV, respectively. Over 99.0% encapsulation efficiency was achieved while preserving payload integrity. The presence of CRISPR plasmids in the ALG NPs was confirmed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. The tests revealed that the nanoparticles were cytocompatible and successfully introduced the Cas9 transgene in HepG2 cells. Nanoparticles-transfected HepG2 was able to edit its target plasmid by introducing double-strand break (DSB) in GFP gene, indicating the bioactivity of CRISPR plasmids encapsulated in alginate nanoparticles. This suggests that this method is suitable for biomedical application in vitro or ex vivo. Future investigation of theses nanoparticles might result in nanocarrier suitable for in vivo delivery of CRISPR/Cas9 system.
    Matched MeSH terms: Transfection
  15. Fulltext PH-responsive strontium nanoparticles for targeted gene therapy against mammary carcinoma cells
    Bakhtiar A, Chowdhury EH
    Asian J Pharm Sci, 2021 Mar;16(2):236-252.
    PMID: 33995617 DOI: 10.1016/j.ajps.2020.11.002
    Genetic intervention via the delivery of functional genes such as plasmid DNA (pDNA) and short-interfering RNA (siRNA) offers a great way to treat many single or multiple genetic defects effectively, including mammary carcinoma. Delivery of naked therapeutic genes or siRNAs is, however, short-lived due to biological clearance by scavenging nucleases and circulating monocytes. Low cellular internalization of negatively-charged nucleic acids further causes low transfection or silencing activity. Development of safe and effectual gene vectors is therefore undeniably crucial to the success of nucleic acid delivery. Inorganic nanoparticles have attracted considerable attention in the recent years due to their high loading capacity and encapsulation activity. Here we introduce strontium salt-based nanoparticles, namely, strontium sulfate, strontium sulfite and strontium fluoride as new inorganic nanocarriers. Generated strontium salt particles were found to be nanosized with high affinity towards negatively-charged pDNA and siRNA. Degradation of the particles was seen with a drop in pH, suggesting their capacity to respond to pH change and undergo dissolution at endosomal pH to release the genetic materials. While the particles are relatively nontoxic towards the cells, siRNA-loaded SrF2 and SrSO3 particles exerted superior transgene expression and knockdown activity of MAPK and AKT, leading to inhibition of their phosphorylation to a distinctive extent in both MCF-7 and 4T1 cells. Strontium salt nanoparticles have thus emerged as a promising tool for applications in cancer gene therapy.
    Matched MeSH terms: Transfection
  16. Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology
    Pahrudin Arrozi A, Shukri SNS, Wan Ngah WZ, Mohd Yusof YA, Ahmad Damanhuri MH, Makpol S
    Appl Biochem Biotechnol, 2017 Nov;183(3):853-866.
    PMID: 28417423 DOI: 10.1007/s12010-017-2468-6
    Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.
    Matched MeSH terms: Transfection
  17. A gain of function p53 gene mutant promotes growth suppression in human liver cancer cells
    Nor Adzimah Johdi, Siti Nurmi Nasir, Rahman Jamal
    Sains Malaysiana, 2017;46:1289-1297.
    Primary liver cancer is one of the most common cancer in the world with highest cancer mortality rate. The most common type of primary liver cancer is hepatocellular carcinoma (HCC). There are many risk factors for liver cancer and currently available treatments for HCC are largely inadequate. Gene mutation and dysfunction of p53 are common and is recognized as an important molecular event in hepatocarcinogenesis. Therefore, replacement of the aberrant p53 gene is an attractive approach in the treatment of HCC providing an alternative treatment for primary HCC. In this study, we assessed whether the transfection with wild-type p53 gene is able to restore the pro-apoptotic effects and evaluate the feasibility of gene therapy in fixing a faulty p53 molecule. We established a non-viral cationic lipid-based p53 gene delivery into two human HCC cell lines namely HLF and PLC/PRF/5 cells. Both cell lines have mutations in the p53 gene. We compared the results with the normal liver cell line, WRL68, that constitutively expresses the wild-type p53 gene. In this study, the introduction of wild-type p53 gene into HLF and PLC/PRF/5 cells resulted in an increased of p53 gene expression, protein expression and cells growth inhibition shown in MTS reduction cell viability assay, FITC-Annexin V and PI apoptosis assay, western blot and caspase activity assay. In summary, the study provides a promising therapeutic approach for p53 gene delivery into HCC patients. The p53 gene delivery can be instituted together with chemotherapy as a combination treatment to induce apoptosis.
    Matched MeSH terms: Transfection
  18. Fulltext Construction of an infectious cDNA clone of Enterovirus 71: insights into the factors ensuring experimental success
    Lazouskaya NV, Palombo EA, Poh CL, Barton PA
    J Virol Methods, 2014 Mar;197:67-76.
    PMID: 24361875 DOI: 10.1016/j.jviromet.2013.12.005
    Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus.
    Matched MeSH terms: Transfection
  19. Fulltext Gene expression profiles in primary duodenal chick cells following transfection with avian influenza virus H5 DNA plasmid encapsulated in silver nanoparticles
    Jazayeri SD, Ideris A, Shameli K, Moeini H, Omar AR
    Int J Nanomedicine, 2013;8:781-90.
    PMID: 23459681 DOI: 10.2147/IJN.S39074
    In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV) that induced cytokine expression, the hemagglutinin (H5) gene of AIV, A/Ck/Malaysia/5858/04 (H5N1) and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5) and formulated using green synthesis of silver nanoparticles (AgNPs) with poly(ethylene glycol) and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL)-18, IL-15, and IL-12β.
    Matched MeSH terms: Transfection/methods*
  20. Fulltext Gene delivery potential of biofunctional carbonate apatite nanoparticles in lungs
    Alhaji SY, Chowdhury EH, Rosli R, Hassan F, Abdullah S
    Biomed Res Int, 2014;2014:646787.
    PMID: 25143941 DOI: 10.1155/2014/646787
    Existing nonviral gene delivery systems to lungs are inefficient and associated with dose limiting toxicity in mammalian cells. Therefore, carbonate apatite (CO3Ap) nanoparticles were examined as an alternative strategy for effective gene delivery to the lungs. This study aimed to (1) assess the gene delivery efficiency of CO3Ap in vitro and in mouse lungs, (2) evaluate the cytotoxicity effect of CO3Ap/pDNA in vitro, and (3) characterize the CO3Ap/pDNA complex formulations. A significantly high level of reporter gene expression was detected from the lung cell line transfected with CO3Ap/pDNA complex prepared in both serum and serum-free medium. Cytotoxicity analysis revealed that the percentage of the viable cells treated with CO3Ap to be almost similar to the untreated cells. Characterization analyses showed that the CO3Ap/pDNA complexes are in a nanometer range with aggregated spherical structures and tended to be more negatively charged. In the lung of mice, highest level of transgene expression was observed when CO3Ap (8 μL) was complexed with 40 μg of pDNA at day 1 after administration. Although massive reduction of gene expression was seen beyond day 1 post administration, the level of expression remained significant throughout the study period.
    Matched MeSH terms: Transfection
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