Screening and evaluation of promoters for effective expression of fluorescent protein and high performance of the modified hyPBase in Zeugodacus cucurbitae.

As non-viral transgenic vectors, the piggyBac transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive piggyBac transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of piggyBac transposase.

Simple SummaryVitellogenin (Vg) is the precursor of the yolk protein gene, which is crucial for insect reproduction. In this study, we identified four Vg genes in Zeugodacus cucurbitae (Coquillett). Their molecular characteristics and expression patterns were analyzed. The four genes are mainly expressed in the fat body tissue of adult female melon flies, and their expression is regulated by the juvenile hormone and ecdysone. Nutritional stress significantly down-regulated their expression, indicating that nutrition-dependent vitellogenic development occurs during ovarian development. RNAi-mediated inhibition of the expression of the four genes resulted in significantly delayed ovarian development in Z. cucurbitae. The results indicate that the four genes play an important role in the development of ovaries in Z. cucurbitae.Vitellogenin (Vg) genes encode the major egg yolk protein precursor in arthropods. In this study, four Vgs were identified in Zeugodacus cucurbitae (Coquillett). Sequence analysis showed that four ZcVgs had the conserved Vg domain. Phylogenetic analysis indicated that four ZcVgs were homologous to the Vgs of Tephritidae insects. The temporal and spatial expression patterns of ZcVgs were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR), and the four ZcVgs showed high expression levels in female adults, especially in the fat body. The expression of ZcVg1 and ZcVg3 was down-regulated by a low dosage (0.5 μg) of 20-hydroxyecdysone (20E), and ZcVg2, ZcVg3, and ZcVg4 were up-regulated by a high dosage (1.0 and 2.0 μg) of 20E. The expression of ZcVg1 and ZcVg2 was up-regulated by 5 μg of juvenile hormone (JH), while all of the ZcVgs were down-regulated by a low and high dosage of JH. Expression of ZcVgs was down-regulated after 24 h of starvation and recovered to normal after nutritional supplementation. After micro-injection of the gene-specific double-stranded RNA, the ZcVgs’ expression was significantly suppressed, and ovarian development was delayed in Z. cucurbitae females. The results indicate that RNA interference of reproduction-related genes is a potential pest control method that works by manipulating female fertility.

In the beginning

Despite much effort, the molecular mechanisms of retinal ganglion cell (RGC) death remain unclear. To identify common cell death-promoting machinery in the mechanically and chemically injured retina, we profiled temporal gene expression patterns and studied their functional roles in the rodent retina. In response to axotomy and intravitreal NMDA injection, 868 genes were commonly differentially expressed compared with those in normal retinas. K-means clustering assigned those common genes to 5 clusters on the basis of their temporal expression patterns, i.e., early, intermediate and late upregulated gene clusters, and early and late downregulated clusters. Most of the common genes and their assigned canonical pathways and molecular functions in each cluster were shared between axotomy and NMDA, indicating that their temporal expression profiles and functional roles are similar. Some of the common genes, including protein tyrosine phosphatases, formed specific molecular networks. Studies using chemical activators/inhibitors and knockout mice have demonstrated that protein tyrosine phosphatase 1/2 and interleukin-1 beta are detrimental to cell survival, whereas endothelin 2, the proteasome and galanin are neuroprotective. Thus, our integrated time-resolved expression profiling of common genes with bioinformatics and functional validation can help us to better understand the precise molecular mechanisms of RGC survival and death.

The genetic control method, which is environmentally friendly and species-specific, has effectively reduced or eliminated pests in many areas. One essential requirement to control a species is the identification of its genetic and molecular elements. Such elements, however, are rarely available in Zeugodacus cucurbitae, a very destructive insect pest worldwide. In this study, we knocked out the white pupae (wp) gene in Z. cucurbitae and generated a wp(-) strain, which has a white pupae phenotype. The white puparium color was successfully restored to brown by inserting the wp gene rescue allele into the genome of the wp(-) strain using piggyBac transgenic technology. The potential wp promoter was then truncated to drive the expression of the wp gene and the puparium color was rescued even by the 605 bp sequence upstream of its transcription initiation site. Further fertility tests showed that knocking out or rescuing the wp gene had no effect on the reproduction of adult flies. In addition, we identified six U6 promoters and tested their promoter activities in the embryos of Z. cucurbitae. The ZcU6-2 and ZcU6-1 promoters exhibited significantly higher activity than the others and are suitable for use in CRISPR technology-based genetic control methods. Our work first shows the success of applying piggyBac transgenic technology in Z. cucurbitae. Our results demonstrate a highly efficient transgenic screening marker by puparium color and the promoter activity of multiple ZcU6 promoters, facilitating the construction of transgenic strains that are used for genetic control of tephritid species. © 2025 Society of Chemical Industry.

The development of Acidithiobacillus ferrooxidans as a non-model host organism for synthetic biology is hampered by a lack of genetic tools and techniques. New plating and liquid-based selection methods were developed to improve the identification of transformed cell lines. Enabled by these methods, a hyperactive transposase was used to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein (sfGFP) gene or a 2-keto decarboxylase (KDC) gene, which enabled the production and secretion of isobutyric acid (IBA). An inverse PCR method was used to identify the insertion sites of the KDC gene in several mutants, leading to the identification of a region on the chromosome that may be suitable for future genetic insertions. These results demonstrate that functional exogenous metabolic genes have been chromosomally integrated into A. ferrooxidans, and this advance will facilitate the future development of these cells for new biotechnology applications. IMPORTANCEAcidithiobacillus ferrooxidans is an iron- and sulfur-oxidizing chemolithoautotroph and is a key member of the microbial consortia used in industrial biomining applications. There is interest in exploiting these cells for other metal recovery applications as well as in developing them as unique nonmodel microbial cell factories. Plasmid-driven expression of exogenous genes has been reported, and homologous recombination has been used to knock out some gene expression. Here, new selection protocols facilitated the development of a transposition method for chromosomal integration of exogenous genes into A. ferrooxidans. This greatly expands the available genetic toolbox, which will open the door to greater metabolic engineering efforts for these cells.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

Pegylated poly-l-arginine derivatives of chitosan for effective delivery of siRNA

Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria have hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonization of, and interaction with the host. In the present study, a range of molecular tools were developed that will allow the closing of some of the gaps in functional analysis of bifidobacteria. A number of promoters were tested for transcriptional activity in Bifidobacterium bifidum S17 using pMDY23, a previously published promoter probe vector. The promoter of the gap gene (Pgap) of B. bifidum S17 yielded the highest promoter activity among the promoters tested. Thus, this promoter and the pMDY23 backbone were used to construct a range of vectors for expression of different fluorescent proteins (FPs). Successful expression of cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry could be shown for three strains representing three different Bifidobacterium spp. The red fluorescent B. bifidum S17/pVG-mCherry was further used to demonstrate application of fluorescent bifidobacteria for adhesion assays and detection in primary human macrophages cultured in vitro. Furthermore, pMGC-mCherry was cloned by combining a chloramphenicol resistance marker and expression of the FP mCherry under the control of Pgap. The chloramphenicol resistance marker of pMGC-mCherry was successfully used to determine gastrointestinal transit time of B. bifidum S17. Moreover, B. bifidum S17/pMGC-mCherry could be detected in fecal samples of mice after oral administration.

Macrophages shine bright

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNAto target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

SOX13 is a member of the SOX family of transcription factors. SOX proteins play essential roles in development, and some are associated with human genetic diseases. SOX13 maps to a multi-disease locus on chromosome 1q31-32, yet its function is unknown. Here we describe the temporal and spatial expression of SOX13 protein during mouse organogenesis. SOX13 is expressed in the three embryonic cell lineages, suggesting that it may direct various developmental processes. SOX13 is expressed in the developing central nervous system including the neural tube and the developing brain. Expression is also detected in the condensing mesenchyme and cartilage progenitor cells during endochondral bone formation in the limb as well as the somite sclerotome and its derivatives. SOX13 is also detected in the developing kidney, pancreas, and liver as well as in the visceral mesoderm of the extra-embryonic yolk sac and spongiotrophoblast layer of the placenta.

BackgroundNoncoding RNAs (ncRNAs) play important roles in a variety of cellular processes. Characterizing the transcriptional activity of ncRNA promoters is therefore a critical step toward understanding the complex cellular roles of ncRNAs.ResultsHere we present an in vivo transcriptional analysis of three C. elegans ncRNA upstream motifs (UM1-3). Transcriptional activity of all three motifs has been demonstrated, and mutational analysis revealed differential contributions of different parts of each motif. We showed that upstream motif 1 (UM1) can drive the expression of green fluorescent protein (GFP), and utilized this for detailed analysis of temporal and spatial expression patterns of 5 SL2 RNAs. Upstream motifs 2 and 3 do not drive GFP expression, and termination at consecutive T runs suggests transcription by RNA polymerase III. The UM2 sequence resembles the tRNA promoter, and is actually embedded within its own short-lived, primary transcript. This is a structure which is also found at a few plant and yeast loci, and may indicate an evolutionarily very old dicistronic transcription pattern in which a tRNA serves as a promoter for an adjacent snoRNA.ConclusionThe study has demonstrated that the three upstream motifs UM1-3 have promoter activity. The UM1 sequence can drive expression of GFP, which allows for the use of UM1::GFP fusion constructs to study temporal-spatial expression patterns of UM1 ncRNA loci. The UM1 loci appear to act in concert with other upstream sequences, whereas the transcriptional activities of the UM2 and UM3 are confined to the motifs themselves.

To gain a better understanding of the natural function of fluorescent proteins, we have undertaken quantitative analyses of these proteins in a single species of coral, Montastraea cavernosa, residing around Turneffe atoll, on the Belizean Barrier Reef. We identified at least 10 members of a fluorescent protein family in this species, which consist of 4 distinct spectral classes. As much as a 10-fold change in the overall expression of fluorescent proteins was observed from specimen to specimen, suggesting that fluorescent proteins are dynamically regulated in response to environmental or physiological conditions. We found that the expression of some proteins was inversely correlated with depth, and that groups of proteins were coordinately expressed. There was no relationship between the expression of fluorescent proteins and the natural coloration of the Montastraea cavernosa specimens in this study. These findings have implications for current hypotheses regarding the properties and natural function of fluorescent proteins.

Zeugodacus cucurbitae is an agricultural pest species with robust reproductive capabilities capable of causing extensive damage. The advent of novel male fertility-related pest control strategies has been an area of active entomological research focused on the sterile insect technique (SIT) strategy. RNA-sequencing analyses were conducted using 16 tissue samples from adult male Z. cucurbitae, leading to the identification of 5338 genes that were differentially expressed between the testes and three other analyzed tissue types. Of these genes, 808 exhibited high levels of testis expression. A quantitative polymerase chain reaction (qPCR) approach was used to validate the expression of ten of these genes selected at random, including ZcTSSK1 and ZcTSSK3, which are testis-specific serine/threonine protein kinase (TSSK) genes. Evaluation via a loss-of-function-based knockdown assay showed that the down-regulation of either of these two genes in males was associated with significantly decreased egg hatching rates. In situ hybridization analyses revealed the expression of both of these transcripts in the transformation zone, and significant decreases in Z. cucurbitae sperm numbers were observed following double-stranded RNA treatment. Together, these results suggested that inhibiting ZcTSSK1 and ZcTSSK3 expression was sufficient to alter male fertility in Z. cucurbitae. These transcriptional sequencing results provide a foundation for further efforts to clarify the regulators of Z. cucurbitae male fertility. These preliminary analyses of the functions of ZcTSSK family genes as regulators of spermatogenesis underscore their importance in the processes integral to male fecundity and their potential as targets for pest control efforts centered on the genetic manipulation of males. © 2023 Society of Chemical Industry.