HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector's replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.
Current viral gene delivery vectors for gene therapy are inefficient due to short-lived transgene expression attributed to the cytosine-phosphate-guanine (CpG) motifs in the transgene. Here we assessed the effects of CpG motif reduction in lentiviral (LV) gene delivery context on the level and duration of reporter gene expression in Chinese Hamster Ovary (CHO) cells, Human Immortalized Myelogenous Leukemia (K562) cells and hematopoietic stem cells (HSCs). The cells were transduced with LV carrying Zero-CpG green fluorescent protein (ZGFP) reporter gene, LV/CMV/ZGFP. The GFP expression was compared to its non CpG-depleted GFP reporter gene LV (LV/CMV/GFP) counterpart. The LV/CMV/ZGFP exhibited prolonged transgene expression in CHO cells and HSCs up to 10 days and 14 days, in the respective cells. This effect was not seen in the transduced K562 cells, which may be due to the DNA hypomethylation status of the cancer cell line. Transgene copy number analysis verified that the GFP expression was not from pseudo-transduction and the transgene remained in the genome of the cells throughout the period of the study. The modest positive effects from the LV/CMV/ZGFP suggest that the reduction of CpG in the LV construct was not substantial to generate higher and more prolonged transgene expression.
Introduction: MiR-3099 was reported to play a role in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development. To further explore its potential regulatory effects on embryonic brain development, this study aims to construct and validate an expression vector of miR-3099 for future gain-of-function and loss-of-function studies. Methods: pCAG-eGFP vector was modified to include IRES2 and miR-3099 with 150bp upstream and downstream genomic sequences. The newly constructed vector, pCAG-miR-3099-IRES2-eGFP, consists of CAG promoter. The in vitro expression level of miR-3099 was measured using stem-loop RT-qPCR after it was transfected into 293FT cell. Later, the vector was electroporated into the embryonic brain at E15.5. Three days later, the E18.5 embryonic brain was harvested and cryopreserved. Immunohistochemistry was performed by using antibody against eGFP to validate the in utero expression of the transgene in the neocortex of the brain. Results: Our finding showed that, the expression level of miR-3099 was significantly upregulated (p
DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.
Adenovirus vector is the most common used vector in clinical gene therapy. The development of adenovirus from the first generation until the helper-dependent adenovirus vector has greatly reduced toxicity and immunogenicity. The helper-dependent adenovirus can also prolong transgene expression. Tissue- or disease-specific approach has been used to improve the specificity of adenoviral vector for cancer gene therapy. This review summarizes some adenoviral gene therapy and targeting approaches available for human cancer as well as animal cancer.
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.
Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.
DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.
Three transgenic HOSUT lines of winter wheat, HOSUT12, HOSUT20, and HOSUT24, each harbor a single copy of the cDNA for the barley sucrose transporter gene HvSUT1 (SUT), which was fused to the barley endosperm-specific Hordein B1 promoter (HO; the HOSUT transgene). Previously, flow cytometry combined with PCR analysis demonstrated that the HOSUT transgene had been integrated into different wheat chromosomes: 7A, 5D, and 4A in HOSUT12, HOSUT20, and HOSUT24, respectively. In order to confirm the chromosomal location of the HOSUT transgene by a cytological approach using wheat aneuploid stocks, we crossed corresponding nullisomic-tetrasomic lines with the three HOSUT lines, namely nullisomic 7A-tetrasomic 7B with HOSUT12, nullisomic 5D-tetrasomic 5B with HOSUT20, and nullisomic 4A-tetrasomic 4B with HOSUT24. We examined the resulting chromosomal constitutions and the presence of the HOSUT transgene in the F2 progeny by means of chromosome banding and PCR. The chromosome banding patterns of the critical chromosomes in the original HOSUT lines showed no difference from those of the corresponding wild type chromosomes. The presence or absence of the critical chromosomes completely corresponded to the presence or absence of the HOSUT transgene in the F2 plants. Investigating telocentric chromosomes occurred in the F2 progeny, which were derived from the respective critical HOSUT chromosomes, we found that the HOSUT transgene was individually integrated on the long arms of chromosomes 4A, 7A, and 5D in the three HOSUT lines. Thus, in this study we verified the chromosomal locations of the transgene, which had previously been determined by flow cytometry, and moreover revealed the chromosome-arm locations of the HOSUT transgene in the HOSUT lines.
Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers.
Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike nonviralmethods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome,which can lead to long-term transgene expression. Lentiviruses have advantages over other types ofviruses due to their capacity to transduce non-dividing cells. An optimized generation of lentivirusescarrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) orCMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by cotransfectinglentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines.Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine(PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvestedat days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFPharvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally,the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transientand stable GFP expression. It was found that the GFP expression declined overtime. However, thetransduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentivirusescarrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells atdifferent efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to proceed to the subsequent analysis of the effect of regulatory elements in future study.
The ability to detect the presence of transgenes in crop-derived foods depends on the quantity and quality of DNA obtained from a product to be analyzed. The efficiency of DNA extraction protocols differs due to the nature of each food product. In this paper, we described two main DNA extraction protocols and their modifications that have been applied and evaluated for DNA extraction from raw and processed food as well as animal feed. The yield and quality for five categories of food and feed samples namely, raw soybean, raw maize, animal feed, smooth tofu and soymilk are discussed. The statistical interaction analyses showed that the cetyltrimethyl ammonium bromide (CTAB) method was proven to be the best method to extract DNA from raw soybean, maize and animal feed samples which not only obtained high DNA yield of 32.7, 28.4 and 33.4 ng DNA/mg sample respectively, but also produced high quality DNA with the absorbance A260/A280 ratio of 1.9, 1.9 and 2.0, respectively. These DNA were suitable for PCR amplification which produced a 164 bp DNA fragment of the lectin gene from soybean, and a 277 bp DNA fragment of the zein gene from maize. In the processed food category, the Wizard isolation method was found to be the best for the extraction of DNA from smooth tofu and soymilk with the yield of 13.2 and 3.4 ng DNA/mg sample, and the quality of the DNA at the absorbance A260/A280 ratio ranged from 1.9 to 1.7. These DNA were successfully amplified using primers specific to the lectin gene of soybean.
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.
Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE-VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.
The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of β-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5'-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.
The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13-20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47-52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.
Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.
The nuclear envelope presents a major barrier to transgene delivery and expression using a non-viral vector. Virus is capable of overcoming the barrier to deliver their genetic materials efficiently into the nucleus by virtue of the specialized protein components with the unique amino acid sequences recognizing cellular nuclear transport machinery. However, considering the safety issues in the clinical gene therapy for treating critical human diseases, non-viral systems are highly promising compared with their viral counterparts. This review summarizes the progress on exploring the nuclear traffic mechanisms for the prominent viral vectors and the technological innovations for the nuclear delivery of non-viral DNA by mimicking those natural processes evolved for the viruses as well as for many cellular proteins.
Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to the cell membrane. Standard microscopy techniques however fall short when finer details including membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM) to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging the invasion complexes in high resolution can provide important insights into the behavior of bacterial vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other molecules.