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  1. Genetic improvement of purslane (Portulaca oleracea L.) and its future prospects
    Amirul Alam M, Juraimi AS, Rafii MY, Hamid AA, Kamal Uddin M, Alam MZ, et al.
    Mol Biol Rep, 2014 Nov;41(11):7395-411.
    PMID: 25085039 DOI: 10.1007/s11033-014-3628-1
    Common purslane (Portulaca oleracea), also known as pigweed, fatweed, pusle, and little hogweed, is an annual succulent herb in the family Portulacaceae that is found in most corners of the globe. From the ancient ages purslane has been treated as a major weed of vegetables as well as other crops. However, worldwide researchers and nutritionists have studied this plant as a potential vegetable crop for humans as well as animals. Purslane is a nutritious vegetable with high antioxidant properties and recently has been recognized as the richest source of α-linolenic acid, essential omega-3 and 6 fatty acids, ascorbic acid, glutathione, α-tocopherol and β-carotene. The lack of vegetable sources of ω-3 fatty acids has resulted in a growing level of attention to introduce purslane as a new cultivated vegetable. In the rapid-revolutionizing worldwide atmosphere, the ability to produce improved planting material appropriate to diverse and varying rising conditions is a supreme precedence. Though various published reports on morphological, physiological, nutritional and medicinal aspects of purslane are available, research on the genetic improvement of this promising vegetable crop are scant. Now it is necessary to conduct research for the genetic improvement of this plant. Genetic improvement of purslane is also a real scientific challenge. Scientific modernization of conventional breeding with the advent of advance biotechnological and molecular approaches such as tissue culture, protoplast fusion, genetic transformation, somatic hybridization, marker-assisted selection, qualitative trait locus mapping, genomics, informatics and various statistical representation have opened up new opportunities of revising the relationship between genetic diversity, agronomic performance and response to breeding for varietal improvement. This review is an attempt to amalgamate the assorted scientific information on purslane propagation, cultivation, varietal improvement, nutrient analyses, medicinal uses and to describe prospective research especially for genetic improvement of this crop.
    Matched MeSH terms: Genetic Engineering/methods*
  2. Short homologies efficiently generate detectable homologous recombination events
    Osahor AN, Tan CY, Sim EU, Lee CW, Narayanan K
    Anal Biochem, 2014 Oct 1;462:26-8.
    PMID: 24929088 DOI: 10.1016/j.ab.2014.05.030
    When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
    Matched MeSH terms: Genetic Engineering/methods*
  3. Heterotrophic cultivation of microalgae for production of biodiesel
    Mohamed MS, Wei LZ, Ariff AB
    Recent Pat Biotechnol, 2011 Aug;5(2):95-107.
    PMID: 21707527
    High cell density cultivation of microalgae via heterotrophic growth mechanism could effectively address the issues of low productivity and operational constraints presently affecting the solar driven biodiesel production. This paper reviews the progress made so far in the development of commercial-scale heterotrophic microalgae cultivation processes. The review also discusses on patentable concepts and innovations disclosed in the past four years with regards to new approaches to microalgal cultivation technique, improvisation on the process flow designs to economically produced biodiesel and genetic manipulation to confer desirable traits leading to much valued high lipid-bearing microalgae strains.
    Matched MeSH terms: Genetic Engineering/methods*
  4. Fulltext Eradication of malaria through genetic engineering: the current situation
    Chong WC, Basir R, Fei YM
    Asian Pac J Trop Med, 2013 Feb;6(2):85-94.
    PMID: 23339908 DOI: 10.1016/S1995-7645(13)60001-2
    Malaria is an intra-cellular parasitic protozoon responsible for millions of deaths annually. Host and parasite genetic factors are crucial in affecting susceptibility to malaria and progression of the disease. Recent increased deployment of vector controls and new artemisinin combination therapies have dramatically reduced the mortality and morbidity of malaria worldwide. However, the gradual emergence of parasite and mosquito resistance has raised alarm regarding the effectiveness of current artemisinin-based therapies. In this review, mechanisms of anti-malarial drug resistance in the Plasmodium parasite and new genetically engineered tools of research priorities are discussed. The complexity of the parasite lifecycle demands novel interventions to achieve global eradication. However, turning laboratory discovered transgenic interventions into functional products entails multiple experimental phases in addition to ethical and safety hurdles. Uncertainty over the regulatory status and public acceptance further discourage the implementation of genetically modified organisms.
    Matched MeSH terms: Genetic Engineering/methods*
  5. The Smart Programmable CRISPR Technology: A Next Generation Genome Editing Tool for Investigators
    Chakraborty C, Teoh SL, Das S
    Curr Drug Targets, 2017;18(14):1653-1663.
    PMID: 27231109 DOI: 10.2174/1389450117666160527142321
    BACKGROUND: The present era is fast experiencing rapid innovation in the genome-editing technology. CRISPR Cas9-mediated targeted genetic manipulation is an easy, cost-effective and scalable method. As a result, it can be used for a broad range of targeted genome engineering.

    OBJECTIVE: The main objective of the present review is to highlight the structural signature, classification, its mechanism and application from basic science to medicine and future challenges for this genome editing tool kit.

    RESULTS: The present review provides a brief description of the recent development of CRISPR-Cas9 genome editing technology. We discuss the paradigms shift for this next generation genome editing technology, CRISPR. The CRISPR structural significance, classification and its different applications are also being discussed. We portray the future challenges for this extraordinary genome in vivo editing tool. We also highlight the role of CRISPR genome editing in curing many diseases.

    CONCLUSION: Scientists and researchers are constantly looking one genome editing tool that is competent, simple and low-cost assembly of nucleases. It can target any particular site without any off-target mutations in the genome. The CRISPR-Cas9 has all of the above characteristics. The genome engineering technology may be a strong and inspiring technology meant for the next generation of drug development.

    Matched MeSH terms: Genetic Engineering/methods*
  6. Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells
    Liew PS, Chen Q, Ng AWR, Chew YC, Ravin NV, Sim EUH, et al.
    Anal Biochem, 2019 10 15;583:113361.
    PMID: 31306622 DOI: 10.1016/j.ab.2019.113361
    Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
    Matched MeSH terms: Genetic Engineering/methods
  7. A Self-Replicating Linear DNA
    Liew PS, Tan TH, Wong YC, Sim EUH, Lee CW, Narayanan K
    ACS Synth Biol, 2020 04 17;9(4):804-813.
    PMID: 32196315 DOI: 10.1021/acssynbio.9b00478
    TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.
    Matched MeSH terms: Genetic Engineering/methods*
  8. Fulltext Evaluation of a Recombinant Newcastle Disease Virus Expressing Human IL12 against Human Breast Cancer
    Mohamed Amin Z, Che Ani MA, Tan SW, Yeap SK, Alitheen NB, Syed Najmuddin SUF, et al.
    Sci Rep, 2019 Sep 30;9(1):13999.
    PMID: 31570732 DOI: 10.1038/s41598-019-50222-z
    The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.
    Matched MeSH terms: Genetic Engineering/methods
  9. Fulltext A review on Lactococcus lactis: from food to factory
    Song AA, In LLA, Lim SHE, Rahim RA
    Microb Cell Fact, 2017 04 04;16(1):55.
    PMID: 28376880 DOI: 10.1186/s12934-017-0669-x
    Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Saccharomyces [corrected] cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.
    Matched MeSH terms: Genetic Engineering/methods*
  10. Escherichia coli tolerance of ultraviolet radiation by in vivo expression of a short peptide designed from late embryogenesis abundant protein
    Huwaidi A, Pathak N, Syahir A, Ikeno S
    Biochem Biophys Res Commun, 2018 09 05;503(2):910-914.
    PMID: 29928878 DOI: 10.1016/j.bbrc.2018.06.095
    Ultraviolet (UV) radiation causes damage in all living organisms, including DNA damage that leads to cell death. Herein, we provide a new technique for UV radiation protection through intracellular short peptide expression. The late embryogenesis abundant (LEA) peptide, which functions as a shield that protects macromolecules from various abiotic stress, was obtained from the Polypedilum vanderplanki group 3 LEA protein. Recombinant Escherichia coli BL21 (DE3) expressing functional LEA short peptide in vivo were exposed to UVA and UVC radiation for 4, 6, and 8 h. E. coli transformants expressing the LEA peptide showed higher cell viability under both UVA and UVC treatment at all time points as compared with that of the control. Furthermore, the cells expressing LEA peptide showed a higher number of colony-forming units per dilution under UVA and UVC treatment. These results suggested that expression of the short peptide could be useful for the development of genetically modified organisms and in applications that require resilience of organisms to UV radiation.
    Matched MeSH terms: Genetic Engineering/methods
  11. Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified
    Rasineni GK, Loh PC, Lim BH
    Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
    PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027
    BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.

    METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.

    RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.

    CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.

    GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

    Matched MeSH terms: Genetic Engineering/methods
  12. Recombineering linear BACs
    Chen Q, Narayanan K
    Methods Mol Biol, 2015;1227:27-54.
    PMID: 25239740 DOI: 10.1007/978-1-4939-1652-8_2
    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
    Matched MeSH terms: Genetic Engineering/methods*
  13. Fulltext Engineering the lactococcal mevalonate pathway for increased sesquiterpene production
    Song AA, Abdullah JO, Abdullah MP, Shafee N, Othman R, Noor NM, et al.
    FEMS Microbiol Lett, 2014 Jun;355(2):177-84.
    PMID: 24828482 DOI: 10.1111/1574-6968.12469
    Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced.
    Matched MeSH terms: Genetic Engineering/methods*
  14. Fulltext Engineering the production of major catechins by Escherichia coli carrying metabolite genes of Camellia sinensis
    Umar KM, Abdulkarim SM, Radu S, Abdul Hamid A, Saari N
    ScientificWorldJournal, 2012;2012:529031.
    PMID: 22645428 DOI: 10.1100/2012/529031
    A mimicked biosynthetic pathway of catechin metabolite genes from C. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. Recombinant E. coli BL (DE3) harbouring the constructs were cultivated for 65 h at 26 °C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (-)-epicatechin (0.01) and (-)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (-)-catechin gallate (0.04 mg/L)) were successfully produced.
    Matched MeSH terms: Genetic Engineering/methods
  15. Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris
    Woon JS, Mackeen MM, Mahadi NM, Illias RM, Abdul Murad AM, Abu Bakar FD
    Biotechnol Appl Biochem, 2016 Sep;63(5):690-698.
    PMID: 26265428 DOI: 10.1002/bab.1431
    The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
    Matched MeSH terms: Genetic Engineering/methods*
  16. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter
    Omidvar V, Siti Nor Akmar A, Marziah M, Maheran AA
    Plant Cell Rep, 2008 Sep;27(9):1451-9.
    PMID: 18563415 DOI: 10.1007/s00299-008-0565-2
    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.
    Matched MeSH terms: Genetic Engineering/methods*
  17. Fulltext Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment
    Rasouli M, Ahmad Z, Omar AR, Allaudin ZN
    BMC Biotechnol, 2011 Nov 03;11:99.
    PMID: 22047106 DOI: 10.1186/1472-6750-11-99
    BACKGROUND: Diabetes mellitus is a complicated disease with a pathophysiology that includes hyperinsulinemia, hyperglycemia and other metabolic impairments leading to many clinical complications. It is necessary to develop appropriate treatments to manage the disease and reduce possible acute and chronic side effects. The advent of gene therapy has generated excitement in the medical world for the possible application of gene therapy in the treatment of diabetes. The glucagon-like peptide-1 (GLP-1) promoter, which is recognised by gut L-cells, is an appealing candidate for gene therapy purposes. The specific properties of L-cells suggest that L-cells and the GLP-1 promoter would be useful for diabetes therapy approaches.

    RESULTS: In this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes.

    CONCLUSION: Our data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents.

    Matched MeSH terms: Genetic Engineering/methods*
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