Transient Gene Expression Studies Research Articles

Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition.

De novo regeneration of Cannabis sativa L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability that need to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies and transformation and genome-editing technologies and open the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplasts have been isolated for transient gene expression studies, but protoplast-to-plant regeneration has not been reported. The present study aims to evaluate the efficacy of using a callus culture system as an abundant tissue source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared with the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0 × 105 protoplasts per mL, cell wall reconstitution and cell division were observed, providing one of the first know reports of cell division from cannabis protoplasts and setting the stage for the future development of a protoplast-to-plant regeneration system.

Development of cell lines from brain, spleen and heart of ornamental blood parrot cichlid and their susceptibility to Tilapia tilapinevirus

Blood parrot cichlid, a hybrid of Amphilophus citrinellus (♂) and Paraneetroplus synspilus (♀), is among the most popular ornamental fishes globally. In this study, three cell lines were established from brain, spleen, and heart tissues of the blood parrot cichlid using explant method and these were designated as parrot fish brain (PFB), parrot fish spleen (PFS) and parrot fish heart (PFH), respectively. All the cell lines have been subcultured for over 120 passages and these grow optimally at 28–32 °C in Leibovitz's L-15 medium containing 20% fetal bovine serum. PFB and PFH cells depict epithelial morphology whereas PFS cells consist of fibroblast-like cells. Although the chromosome number of blood parrot cichlid was 2n = 48, the cell lines exhibited heteroploidy and aneuploidy, suggesting transformation. The sequencing of amplified fragments of two mitochondrial genes, namely COI and 16S rRNA confirmed that the cell lines originated from blood parrot cichlid. The three cell lines were free of Mycoplasma contamination. Besides, their successful transfection with a GFP reporter gene suggested potential application in transient gene expression studies. All the three cell lines were susceptible to Tilapia tilapinevirus, also known as Tilapia lake virus (TiLV), which was indicated by appearance of cytopathic effects and RT-PCR. Of the three cell lines, PFS was found to be the most permissive with maximum TiLV copy number of 2.5 × 108 per 100 ng of total RNA, as determined by qRT-PCR. Importantly, the TiLV disease was successfully reproduced in naive tilapia after injection of cell culture supernatant from TiLV-infected PFS cell line, and this was evidenced by clinical signs, histopathological alterations, reisolation of virus and specific amplification in RT-PCR. The three newly established cell lines were submitted to National Repository on Fish Cell Lines at ICAR-NBFGR. This forms the first report of development of cell lines from blood parrot cichlid, and these would be useful for surveillance of diseases suspected to be of viral origin in this candidate species and also other ornamental cichlids.

Protoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.

Cannabis sativa L. is a valuable, up-and-coming industrial crop with a substantially growing market. However, due to an extended period of legal restriction, research with cannabis has been limited, particularly in laboratory settings. Expanding the application of biotechnological techniques to cannabis can facilitate addressing species-specific impediments to improving crop traits and further fundamental understanding of its intricacies. Here, we describe application of protoplast transformation for the study of transient gene expression in a low-THC cannabis cultivar. To produce explant tissue as a source of protoplasts, a method for hormone-free in vitro micropropagation is established. Protoplasts are isolated from young leaves of the micropropagated stocks and transiently transformed with plasmid DNA carrying a fluorescent marker gene. This is the first report of protoplast transformation in this species. A protoplast isolation yield is achieved of up to 2 × 106 cells per gram of leaf material, vitality staining shows that up to 82 % of isolated protoplasts are viable, and quantification of the cells expressing a fluorescent protein indicates that up to 31 % of the cells can be successfully transformed. Additionally, protoplasts are transformed with an auxin-responsive reporter gene and the reaction to treatment with indole-3-acetic acid is quantified using flow cytometry. This work demonstrates that relatively minor modification of standard techniques can be used to study this important emerging crop.

A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus

BackgroundThe study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava’s recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L.MethodsCassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey’s HSD test and the 2−ΔΔCTstatistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model.ResultsThe differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces.ConclusionsThis protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

A Highly Efficient Agrobacterium-Mediated Method for Transient Gene Expression and Functional Studies in Multiple Plant Species

Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein–protein interactions in the sucrose biosynthesis pathway in Arabidopsis.

Early Studies on Protoplast Isolation of Ludisia discolor, A Wild Orchid.

The terrestrial Ludisia discolor, also referred to as the jewel orchid is prized for the quality of its leaves. L. discolor is known as a medicinal herb and is touted for its heat- and pathogen-resisting qualities. L. discolor is valuable in the production of both flavonoids and anthocyanins, antioxidants that are exalted in the health industry. Plant cell cultures have emerged as alternative sources of anthocyanin production. Plant protoplast cultures are used frequently in transient gene expression studies and in the establishment of callus and cell suspension cultures. Benefits of plant protoplast system include similarity to cells found in plant tissues, reproduction under controlled conditions, and prevention of masking of stress responses to previous handling techniques. A study was conducted to assess the amenability of the stem and leaves of L. discolor to protoplast isolation. The stem and leaf segments were weighed, sliced into thin layers, immersed in a digestion medium, washed and then cultured onto a recovery medium. Results indicated that the production of plant protoplasts from L. discolor may be viewed as an alternative in the generation of cell cultures and ultimately in the production of anthocyanins from the cell cultures.

Comparison of procedures for DNA coating of micro-carriers in the transient and stable biolistic transformation of sugarcane

Biolistic gene transfer is one of the preferred methods for both transient gene expression studies and stable genetic transformation for improvement of crops including sugarcane. In preparation for bombardment, DNA is precipitated onto micro-particles in the presence of a cationic polyamine and buffer. Three different DNA coating procedures were compared for biolistic transformation of sugarcane using linear expression cassettes: spermidine (free base), protamine sulfate, and the Seashell DNAdel™ Gold Carrier with proprietary precipitation buffer from Seashell Technology. All three DNA coating procedures produced transient and stable sugarcane transformation events in multiple experiments. Spermidine resulted in three-fold and 2.65-fold higher transient GUS expression than protamine and Seashell DNAdel™, respectively when averaged over 10 coating reactions per treatment. However, no significant difference in stable genetic transformation was found between the three DNA coating procedures. This study determined that spermidine-, protamine-, and Seashell DNAdel™ Gold Carrier-mediated DNA coating can be equally effective for biolistic transformation of sugarcane.

PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process

Polyethylenimine (PEI) has been used widely in transient gene expression studies of mammalian cells. We performed transient gene expression in suspension Chinese hamster ovary cells using a one-step transfection procedure in which DNA and PEI were simultaneously added to a cell culture in suspension without prior PEI/DNA complex incubation. To further understand the effect of PEI/DNA formation on the transfection and expression of exogenous gene in shaking state, we investigated the diameter and overcharge of the PEI/DNA complex. The results showed that the diameter of the complex was smaller with more positive charge when the PEI/DNA ratio was higher. Moreover, DNA more easily penetrated cells and nuclei at higher PEI concentrations. The highest transcription level, transfection efficiency, and GFP expression were obtained when the PEI/DNA ratio was 5:1.

Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

BackgroundTransient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue.ResultsHere, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima). In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days.ConclusionsThe simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

Histone Deacetylase 9 Activates γ-Globin Gene Expression in Primary Erythroid Cells

Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies probably involve protein modifications by histone deacetylases (HDACs) that mediate γ-globin gene regulation. However, the role of individual HDACs in globin gene expression is not very well understood; thus, the focus of our study was to identify HDACs involved in γ-globin activation. K562 erythroleukemia cells treated with the HbF inducers hemin, trichostatin A, and sodium butyrate had significantly reduced mRNA levels of HDAC9 and its splice variant histone deacetylase-related protein. Subsequently, HDAC9 gene knockdown produced dose-dependent γ-globin gene silencing over an 80-320 nm range. Enforced expression with the pTarget-HDAC9 vector produced a dose-dependent 2.5-fold increase in γ-globin mRNA (p < 0.05). Furthermore, ChIP assays showed HDAC9 binding in vivo in the upstream Gγ-globin gene promoter region. To determine the physiological relevance of these findings, human primary erythroid progenitors were treated with HDAC9 siRNA; we observed 40 and 60% γ-globin gene silencing in day 11 (early) and day 28 (late) progenitors. Moreover, enforced HDAC9 expression increased γ-globin mRNA levels by 2.5-fold with a simultaneous 7-fold increase in HbF. Collectively, these data support a positive role for HDAC9 in γ-globin gene regulation.

Molecular Characterization of a Salt‐Inducible Monodehydroascorbate Reductase from the Halophyte Avicennia marina

Salinity poses a major threat to crop productivity. Our earlier work has used the halophytic plant Avicennia marina as a model organism for mining genes that function in salinity stress tolerance. Here we report the isolation and characterization of a monodehydroascorbate reductase (MDAR) from this plant. MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for reactive oxygen species scavenging. A cDNA clone encoding MDAR was isolated from a cDNA library created from a salt‐stressed leaf of A. marina. A transit peptide at the N‐terminal region of Am‐MDAR suggested chloroplastic localization. Transcript profiling for Am‐MDAR revealed that the gene is expressed in response to salinity and oxidative stress (high‐intensity light, H2O2, and iron overload). The genomic clone of Am‐MDAR contained 16 exons. The presence of two identical MDAR transcripts, with and without exon 3, indicated possible exon skipping. A 1167‐bp fragment corresponding to the 5′ upstream region of Am‐MDAR was isolated, and transient reporter gene expression studies revealed it to be a functional promoter. In‐silico analysis of this sequence revealed the presence of putative light‐ and abiotic‐stress regulatory elements. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.

Characterization of an Oxidative Stress Inducible Nonspecific Lipid Transfer Protein Coding cDNA and its Promoter from Drought Tolerant Plant Prosopis juliflora

Abiotic stress is the principal cause of crop failure worldwide. Prosopis juliflora is a hardy plant reported to be tolerant to drought, salinity, extremes of soil pH, and heavy metal stress. Here, we report the characterization of a cDNA clone for a putative nonspecific lipid transfer protein (Pj LTP1) found abundantly in a drought stressed leaf cDNA library of P. juliflora and its promoter. A multiple sequence analysis of Pj LTP1 with well-established and officially designated nonspecific lipid transfer proteins allergens revealed high similarity at the amino acid level. Northern analysis of Pj LTP1 in P. juliflora leaves under oxidative stress revealed steady upregulation at the time points analyzed. A 929-bp fragment was isolated from the 5′ end of Pj LTP1, and transient reporter gene expression studies revealed it to be a functional promoter. Several cis-acting elements previously reported to function in stress response were found in this promoter. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.

Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression

In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.

Transformation of Moss Physcomitrella patens Gametophytes Using a Biolistic Projectile Delivery System

INTRODUCTIONThis method makes use of biolistic bombardment to propel gold particles coated with DNA into protonemal tissue of the moss Physcomitrella patens. It is especially suitable for transient gene expression studies, but it can be used to obtain stable transformants.

Differential regulation of zebrafish metallothionein-II (zMT-II) gene transcription in ZFL and SJD cell lines by metal ions

Two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5′-flanking region (1514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) and caudal fin (SJD) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn 2+ and that this Zn 2+ induction was dose-dependent. Further transient gene expression assay of the zMT-IIA gene promoter was carried out to study the effects of various metal ions (Zn 2+, Cd 2+, Cu 2+, Hg +, As 3+, As 5+, Cr 3+ and Cr 6+), and Zn 2+ and Cd 2+ were found to be the most efficient MT gene inducers of zMT-II. As 3+ was a weak inducer of zMT-II in the two cell lines, and Hg + caused significant induction only in the SJD cells. No significant induction was found in the other metal ion exposures. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn 2+ or Cd 2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein.

Transient gene expression in guard cell chloroplasts of tobacco using ArF excimer laser microablation

In this paper, we report a novel method for delivering genes into chloroplasts of tobacco cells using laser microablation. The plasmid pLD200-GFP was introduced into chloroplasts of Nicotiana tabacum cv. Xanthi guard cells and transient GFP expression was detected in the chloroplasts after 2-3 d of incubation. The technique uses an argon fluoride (ArF) excimer laser to perforate the cell surface in a 4 mum(2) area in the presence of plasmid coated gold microparticles. Pretreatment of guard cells to promote stomatal closure prior to laser ablation resulted in a significant increase in the survival rate of cells and a transient expression rate of 2-3% in trial number basis was archived. Our method has unique advantages such as avoiding laborious pretreatments that adversely affect cell viability and specific delivery of transgenes into a desired cell in complex leaf tissue. This technique is a potential tool for cell specific transient gene expression studies for elucidation of gene regulation and expression.

Cis-Acting Gene Regulatory Activities in the Terminal Regions ofSleeping BeautyDNA Transposon-Based Vectors

Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of nonviral integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles. Because of concerns related to mutagenesis and/or activation of cellular genes by integrating vectors, much attention has been paid to integration site preferences and the ability of vectors to influence expression of neighboring genes. Here, we test the hypothesis that terminal repeats of transposons carry cis-acting regulatory sequences. In transient gene expression studies, we demonstrate that the inverted repeats of SB direct gene expression in HeLa cells to levels that are 3-fold higher than in promoter-deficient controls. Inverted repeats pointing toward the transposon center consistently facilitate the highest levels of activity in a number of cell lines. We show that transposon sequences flanking the inverted repeats of SB are required for positive effects on gene expression and, moreover, that these regions contain both stimulatory and inhibitory cis-acting elements. In the context of an integrated SB vector the regulatory activities of the transposon termini are sufficient to drive expression of selectable marker genes carried by the transposon, indicating that opposing transcriptional activities originating from the transposon termini may influence expression of its genetic cargo. Finally, detection of regulatory properties of the terminal repeats of the active Tc3 element from Caenorhabditis elegans leads to the suggestion that transcriptional activities of the inverted repeats are conserved among Tc1/mariner transposons in nature. Our data suggest that SB-based gene vectors may carry ancient properties of self-regulation with potential relevance for SB-directed therapeutic gene transfer.

Hepatic Gene Expression Changes in Hypothyroid Juvenile Mice: Characterization of a Novel Negative Thyroid-Responsive Element

The molecular mechanisms involved in the response of developing mice to disruptions in maternal thyroid hormone (TH) homeostasis are poorly characterized. We used DNA microarrays to examine a broad spectrum of genes from the livers of mice rendered hypothyroid by treating pregnant mice from gestational d 13 to postnatal d 15 with 6-propyl-2-thiouracil in drinking water. Twenty-four individuals (one male and one female pup from six litters of control or 6-propyl-2-thiouracil treatment groups, respectively) were profiled using Agilent oligonucleotide microarrays. MAANOVA identified 96 differentially expressed genes (false discovery rate adjusted P < 0.1 and fold change > 2 in at least one gender). Of these, 72 genes encode proteins of known function, 15 of which had previously been identified as regulated by TH. Pathway analysis revealed these genes are involved in metabolism, development, cell proliferation, apoptosis, and signal transduction. An immediate-early response gene, Nr4a1 (nuclear receptor subfamily 4, group A, member 1), was up-regulated by 3-fold in hypothyroid juvenile mouse liver; treatment of HepG2 cells with T(3) resulted in down-regulation of Nr4a1. A potential thyroid response element -1218 to -1188 bp upstream of the promoter region of Nr4a1 was identified and demonstrated to bind TH receptor (TR)-alpha and TRbeta. Point mutation or deletion of the sequence containing the potential Nr4a1-thyroid response element in transient gene expression studies resulted in both higher basal expression and loss of T(3) regulatory capacity, suggesting that this site is responsible for the negative regulation of gene expression by TR and TH.

Transient Expression in Protoplasts

INTRODUCTIONThe study of transient gene expression provides a useful complement to the study of stably transformed plants. Transient assays offer a quick method of testing the effects of genes, using either phenotypic, molecular, or biochemical readouts. Transient assays based on Agrobacterium-mediated transformation of leaf explants have been described for other plant species, but it is not known how well these assays work in Arabidopsis. Alternatives include transient expression in protoplasts or in regenerating roots. The latter is especially suitable for investigating root-specific gene function.

Transfection of insect cell lines using polyethylenimine.

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85-90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.