Displaying publications 1 - 20 of 60 in total

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  1. 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*
  2. A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles
    Feng S, Xie X, Liu J, Li A, Wang Q, Guo D, et al.
    J Nanobiotechnology, 2023 Oct 10;21(1):370.
    PMID: 37817254 DOI: 10.1186/s12951-023-02139-z
    Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.
    Matched MeSH terms: Genetic Engineering
  3. 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*
  4. 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*
  5. Achieving crop stress tolerance and improvement--an overview of genomic techniques
    Rasool S, Ahmad P, Rehman MU, Arif A, Anjum NA
    Appl Biochem Biotechnol, 2015 Dec;177(7):1395-408.
    PMID: 26440315 DOI: 10.1007/s12010-015-1830-9
    The inexorable exposure of plants to the combinations of abiotic stresses has affected the worldwide food supply. The crop improvement against these abiotic stresses has been captivating approach to increase the yield and enhance the stress tolerance. By using traditional and modern breeding methods, the characters that confer tolerance to these stresses were accomplished. No doubt genetic engineering and molecular breeding have helped in comprehending the intricate nature of stress response. Understanding of abiotic stress-involved cellular pathways provides vital information on such responses. On the other hand, genomic research for crop improvement has raised new assessments in breeding new varieties against abiotic stresses. Interpretation of responses of the crop plants under stress is of great significance by studying the main role of crops in food and biofuel production. This review presents genomic-based approaches revealing the complex networks controlling the mechanisms of abiotic stress tolerance, and the possible modes of assimilating information attained by genomic-based approaches due to the advancement in isolation and functional analysis of genes controlling the yield and abiotic stress tolerance are discussed.
    Matched MeSH terms: Genetic Engineering
  6. Fulltext Advances in Genetic Improvement for Tocotrienol Production: A Review
    Babura SR, Abdullah SNA, Khaza Ai H
    J Nutr Sci Vitaminol (Tokyo), 2017;63(4):215-221.
    PMID: 28978868 DOI: 10.3177/jnsv.63.215
    Tocotrienols are forms of vitamin E that are present in several important food crops. Compared to tocopherols, less research has been conducted on these compounds because of their low bioavailability and distribution in plant tissues. Both tocotrienols and tocopherols are known for their antioxidant and anticancer activities, which are beneficial for both humans and animals. Moreover, tocotrienols possess certain properties which are not found in tocopherols, such as neuroprotective and cholesterol-lowering activities. The contents of tocotrienols in plants vary. Tocotrienols constitute more than 70% and tocopherols less than 30% of the total vitamin E content in palm oil, which is the best source of vitamin E. Accumulation of tocotrienols also occurs in non-photosynthetic tissues, such as the seeds, fruits and latex of some monocotyledonous and dicotyledonous plant species. The use of biotechnological techniques to increase the tocotrienol content in plants, their biological functions, and benefits to human health are discussed in this review.
    Matched MeSH terms: Genetic Engineering
  7. Advances to improve the eating and cooking qualities of rice by marker-assisted breeding
    Phing Lau WC, Latif MA, Y Rafii M, Ismail MR, Puteh A
    Crit Rev Biotechnol, 2016;36(1):87-98.
    PMID: 24937109 DOI: 10.3109/07388551.2014.923987
    The eating and cooking qualities of rice are heavily emphasized in breeding programs because they determine market values and they are the appealing attributes sought by consumers. Conventional breeding has developed traditional varieties with improved eating and cooking qualities. Recently, intensive genetic studies have pinpointed the genes that control eating and cooking quality traits. Advances in genetic studies have developed molecular techniques, thereby allowing marker-assisted breeding (MAB) for improved eating and cooking qualities in rice. MAB has gained the attention of rice breeders for the advantages it can offer that conventional breeding cannot. There have been successful cases of using MAB to improve the eating and cooking qualities in rice over the years. Nevertheless, MAB should be applied cautiously given the intensive effort needed for genotyping. Perspectives from conventional breeding to marker-assisted breeding will be discussed in this review for the advancement of the eating and cooking qualities of fragrance, amylose content (AC), gel consistency (GC) and gelatinization temperature (GT) in rice. These four parameters are associated with eating and cooking qualities in rice. The genetic basis of these four parameters is also included in this review. MAB is another approach to rice variety improvement and development in addition to being an alternative to genetic engineering. The MAB approach shortens the varietal development time, and is therefore able to deliver improved rice varieties to farmers within a shorter period of time.
    Matched MeSH terms: Genetic Engineering
  8. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) by recombinant Escherichia coli expressing leucine metabolism-related enzymes derived from Clostridium difficile
    Saika A, Watanabe Y, Sudesh K, Tsuge T
    J Biosci Bioeng, 2014 Jun;117(6):670-5.
    PMID: 24484910 DOI: 10.1016/j.jbiosc.2013.12.006
    An obligate anaerobic bacterium Clostridium difficile has a unique metabolic pathway to convert leucine to 4-methylvalerate, in which 4-methyl-2-pentenoyl-CoA (4M2PE-CoA) is an intermediate of this pathway. 4M2PE-CoA is also able to be converted to 3-hydroxy-4-methylvalerate (3H4MV), a branched side chain monomer unit, for synthesis of polyhydroxyalkanoate (PHA) copolymer. In this study, to synthesize 3H4MV-containing PHA copolymer from leucine, the leucine metabolism-related enzymes (LdhA and HadAIBC) derived from C. difficile and PHA biosynthesis enzymes (PhaPCJAc and PhaABRe) derived from Aeromonas caviae and Ralstonia eutropha were co-expressed in the codon usage-improved Escherichia coli. Under microaerobic culture conditions, this E. coli was able to synthesize P(3HB-co-12.2 mol% 3H4MV) from glucose with the supplementation of 1 g/L leucine. This strain also produced P(3HB-co-12.6 mol% 3H4MV) using the culture supernatant of leucine overproducer E. coli strain NS1391 as the medium for PHA production, achieving 3H4MV copolymer synthesis only from glucose. Furthermore, we tested the feasibility of the 3H4MV copolymer synthesis in E. coli strain NS1391 from glucose. The recombinant E. coli NS1391 was able to synthesize P(3HB-co-3.0 mol% 3H4MV) from glucose without any leucine supplementation. This study demonstrates the potential of the new metabolic pathway for 3H4MV synthesis using leucine metabolism-related enzymes from C. difficile.
    Matched MeSH terms: Genetic Engineering
  9. Fulltext Characterisation of Drosophila Ubx CPTI000601 and hth CPTI000378 protein trap lines
    Choo SW, Beh CY, Russell S, White R
    ScientificWorldJournal, 2014;2014:191535.
    PMID: 25389534 DOI: 10.1155/2014/191535
    In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The Ubx (CPTI000601) line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hth (CPTI000378) is completely lethal as a homozygote, the hth (CPTI000378) /hth (C1) genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hth (CPTI000378) /Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hth (CPTI000378), hth (CPTI000378) /hth (C1), or hth (CPTI000378) /Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins.
    Matched MeSH terms: Genetic Engineering
  10. 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
  11. Fulltext Comparison of invasion by human microvascular endothelial cell lines in response to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in a threedimensional (3D) cell culture system
    Ng CT, Yip WK, Mohtarrudin N, Seow HF
    Malays J Pathol, 2015 Dec;37(3):219-25.
    PMID: 26712666 MyJurnal
    Immortalized human endothelial cells are widely used as in vitro models for debilitating conditions such as cancer, cardiovascular and ocular diseases. Human microvascular endothelial cell (HMEC-1) is immortalized via stable transfection with a gene encoding SV40 large antigen whilst telomerase-immortalized human microvascular endothelial (TIME) cells is immortalized by engineering the human telomerase catalytic protein (hTERT) into primary microvascular endothelial cells. Here, we established a three-dimensional (3D) spheroid invasion assay with HMEC-1 and TIME and compared the difference in their ability to invade through the collagen matrix in response to exogenous growth factors, namely vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
    Matched MeSH terms: Genetic Engineering
  12. Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter
    Masani MY, Parveez GK, Izawati AM, Lan CP, Siti Nor Akmar A
    Plasmid, 2009 Nov;62(3):191-200.
    PMID: 19699761 DOI: 10.1016/j.plasmid.2009.08.002
    One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (beta-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (beta-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.
    Matched MeSH terms: Genetic Engineering
  13. Fulltext Cyanobacteria: Photoautotrophic Microbial Factories for the Sustainable Synthesis of Industrial Products
    Lau NS, Matsui M, Abdullah AA
    Biomed Res Int, 2015;2015:754934.
    PMID: 26199945 DOI: 10.1155/2015/754934
    Cyanobacteria are widely distributed Gram-negative bacteria with a long evolutionary history and the only prokaryotes that perform plant-like oxygenic photosynthesis. Cyanobacteria possess several advantages as hosts for biotechnological applications, including simple growth requirements, ease of genetic manipulation, and attractive platforms for carbon neutral production process. The use of photosynthetic cyanobacteria to directly convert carbon dioxide to biofuels is an emerging area of interest. Equipped with the ability to degrade environmental pollutants and remove heavy metals, cyanobacteria are promising tools for bioremediation and wastewater treatment. Cyanobacteria are characterized by the ability to produce a spectrum of bioactive compounds with antibacterial, antifungal, antiviral, and antialgal properties that are of pharmaceutical and agricultural significance. Several strains of cyanobacteria are also sources of high-value chemicals, for example, pigments, vitamins, and enzymes. Recent advances in biotechnological approaches have facilitated researches directed towards maximizing the production of desired products in cyanobacteria and realizing the potential of these bacteria for various industrial applications. In this review, the potential of cyanobacteria as sources of energy, bioactive compounds, high-value chemicals, and tools for aquatic bioremediation and recent progress in engineering cyanobacteria for these bioindustrial applications are discussed.
    Matched MeSH terms: Genetic Engineering*
  14. Dissolving the engineering moral dilemmas within the Islamic ethico-legal praxes
    Solihu AK, Ambali AR
    Sci Eng Ethics, 2011 Mar;17(1):133-47.
    PMID: 19937149 DOI: 10.1007/s11948-009-9185-7
    The goal of responsible engineers is the creation of useful and safe technological products and commitment to public health, while respecting the autonomy of the clients and the public. Because engineers often face moral dilemma to resolve such issues, different engineers have chosen different course of actions depending on their respective moral value orientations. Islam provides a value-based mechanism rooted in the Maqasid al-Shari'ah (the objectives of Islamic law). This mechanism prioritizes some values over others and could help resolve the moral dilemmas faced in engineering. This paper introduces the Islamic interpretive-evaluative maxims to two core issues in engineering ethics: genetically modified foods and whistleblowing. The study aims primarily to provide problem-solving maxims within the Maqasid al-Shari'ah matrix through which such moral dilemmas in science and engineering could be studied and resolved.
    Matched MeSH terms: Genetic Engineering/ethics
  15. Fulltext Elevated CO2 improves both lipid accumulation and growth rate in the glucose-6-phosphate dehydrogenase engineered Phaeodactylum tricornutum
    Wu S, Gu W, Huang A, Li Y, Kumar M, Lim PE, et al.
    Microb Cell Fact, 2019 Sep 23;18(1):161.
    PMID: 31547820 DOI: 10.1186/s12934-019-1214-x
    BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation.

    RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains.

    CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.

    Matched MeSH terms: Genetic Engineering
  16. Fulltext Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
    Nyon MP, Du L, Tseng CK, Seid CA, Pollet J, Naceanceno KS, et al.
    Vaccine, 2018 03 27;36(14):1853-1862.
    PMID: 29496347 DOI: 10.1016/j.vaccine.2018.02.065
    Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377-588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.
    Matched MeSH terms: Genetic Engineering*
  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*
  18. Fulltext Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
    Koko I, Song AA, Masarudin MJ, Abdul Rahim R
    BMC Biotechnol, 2019 11 27;19(1):82.
    PMID: 31775775 DOI: 10.1186/s12896-019-0575-x
    BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system.

    RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.

    CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

    Matched MeSH terms: Genetic Engineering
  19. 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*
  20. 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
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