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PubMed2026

A stable and potent buffalo EF1α1 promoter for robust gene expression in mammalian systems.

This study reports the first isolation and characterization of the buffalo EF1α1 promoter, demonstrating its strong gene expression activity both in vitro across diverse cultured cell types and in vivo across multiple mouse organs. Although viral promoters, such as cytomegalovirus (CMV) and simian virus (SV40), are widely used for their strong expression in various cell lines in mammalian expression systems and in animal tissues, they are prone to methylation-induced transcriptional silencing and subsequent loss of exogenous gene expression. The most effective alternative to viral promoters is the synthetic hybrid CAG promoter (cytomegalovirus major immediate-early enhancer combined with the chicken beta-actin promoter) or mammalian cellular promoter such as human elongation factor 1 alpha (hEF1α), which drives strong gene expression but lacks consistency and is limited in their in vivo expression potential due to their vulnerability to epigenetic silencing. To overcome these challenges, the bbEF1α1 promoter was cloned and evaluated both in vitro and in vivo. It consistently drives higher levels of exogenous gene expression than CMV in diverse cell lines. Importantly, transgene expression was achieved in various organs of transgenic mice and in muscle tissue following in vivo electroporation. These findings establish the bbEF1α1 promoter as a powerful ubiquitous driver of gene expression, offering high stability with broad applications in gene therapy, biopharmaceutical production, and functional genomics.

Animals↗

PubMed2026

Functional analysis of stem-loop structures within the SARS-CoV-2 5' untranslated region using a plasmid-based reporter system.

The 5' untranslated region (5'UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains highly conserved stem-loop structures that regulate viral gene expression. This study investigated the functional contributions of selected 5'UTR stem-loop elements to reporter gene expression using a plasmid-based mammalian expression system. Five constructs were tested using a non-integrating plasmid: the wild-type (WT) 5'UTR fused to GFP under the CMV promoter, and four deletion variants (ΔB, ΔC, ΔD, and ΔE) corresponding to deletions of stem-loop 4 (SL4), SL4.5, SL5, and SL5a, respectively. Following transfection into HEK293 cells, GFP fluorescence was quantified using a fluorescence microplate reader, and relative GFP transcript abundance was assessed by RT-qPCR. Deletion of SL4 (ΔB) resulted in marked reduction in both fluorescence and relative transcript abundance compared to WT construct, indicating substantially reduced reporter gene expression. In contrast, deletion of SL4.5, SL5, or SL5a did not produce the pronounced reduction observed for ΔB, although descriptive RT-qPCR analysis indicated differences in relative transcript abundance among these variants. Statistical analysis of fluorescence data demonstrated significant differences among constructs (one-way ANOVA, p < 0.05). Because the reporter assay was based on plasmid expression, the observed differences likely reflect combined contributions from transcription, transcript abundance, RNA stability, and translation rather than translation alone. These findings demonstrate that the SL4 region contributes substantially to reporter gene expression in this experimental system, whereas the remaining stem-loop regions examined exert comparatively modest effects. This study provides additional insight into the functional organization of the SARS-CoV-2 5'UTR and establishes a framework for future investigations aimed at distinguished the transcriptional, post-transcriptional, and translational contributions of individual RNA structural elements.

5' Untranslated Regions↗

PubMed2026

A CHO-Derived Matrix Attachment Region Enhances Transgene Dosage, SATB1 Recruitment, and Monoclonal Antibody Expression in a Dual-Promoter Vector System.

The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.

Animals↗

PubMed2025

Construction and Isolation of Recombinant Vaccinia Virus by Homologous Recombination Using Fluorescent Protein Markers.

Genetic modification of vaccinia virus (VACV) is a fundamental and valuable research technique in elucidating the function of VACV genes, as well as the development as vaccine vectors for other infectious diseases, oncolytic therapeutics for cancers, and protein expression systems in mammalian cells. Because of the large size of poxvirus genome and noninfectious feature of the naked viral DNA, construction of recombinant VACV relies on intracellular homologous recombination between transfected DNA and replicating viral DNA in infected cells occurred in VACV infected cells. The efficiency of homologous recombination event for vaccinia virus is relatively low, and recombinant viruses only account for 0.1% of progeny viruses. Therefore, fluorescent protein markers are often included in the transfected DNA to facilitate the selection and screening of recombined viruses. Here we provide a detailed procedure for the design, generation, isolation, and detection of recombinant VACV by homologous recombination using fluorescent protein markers.

Vaccinia virus↗

PubMed1979

Detecting mutations expressed during early development of cultured mammalian embryos.

Mammalian embryo culture systems can be used to study dominant lethal, recessive lethal, and visible mutations that are expressed between fertilization and early stages of organogenesis. The incidence of dominant lethal mutations has been determined for cultured mouse embryos by morphological observations during pre-implantation and early post-implantation growth in vitro, and the mechanisms of dominant lethal mutations have been studied cytogenetically. Recessive lethal mutations have been studied with cultured embryos, although they can be initially detected only with appropriate breeding protocols. Visible mutations that are due to deletions or single-site base alterations in the DNA can be detected in embryos by isoelectric focusing and gel-electrophoresis techniques.--The principal advantage of cultured embryos for detecting mutations and studying the mechanism of action of particular mutagens is that embryos are accessible for analysis before death with a minimum of indirect maternal effects. The primary disadvantages, which may be alleviated with improved culture conditions, are that only a limited amount of tissue is available, attrition and retardation occur, and offspring are not recovered for further breeding studies.

Animals↗

PubMed1975

Gene transfer to human cells: transducing phage lambda plac gene expression in GMI-gangliosidosis fibroblasts.

Genetic information from the bacterium Escherichia coli was transferred to human cells by means of the specialized transducing phage lambda plac carrying the bacterial z gene for the enzyme beta-galactosidase (geta-D-galactoside galactohydrolase, EC 3.2.1.23). As recipient cells, cultured skin fibroblasts from a patient with generalized gangliosidosis (GMI-gangliosidosis Type I) characterized by a severe deficiency of beta-galactosidase activity were used. The deficient human cells were incubated with the bacteriophage lambda plac or lambda plac DNA and beta-galactosidase activity was measured in order to detect gene transfer and acceptance of the prokaryotic information in the mammalian system for transcription and translation. The expression of the phage genome in the deficient fibroblasts could be demonstrated by detection of higher beta-galactosidase activity after incubation with phage lambda plac in three out of 19 experiments and in four out of 16 experiments after treatment with lambda plac DNA. Lambda plac DNA induced much higher enzyme activities than infective phage particles. Immunochemical and physicochemical assays could not distinguish the induced beta-galactosidase activity from that of the z-gene product of E. coli.

Cell Line↗

PubMed1978

Growth dependence of phosphoglyceric acid dehydrogenase activity in cultured rat liver cells.

Rat liver epithelial cells in culture (WIRL-3C) have the enzymes that synthesize serine from 3-phophoglyceric acid. Both phosphoglyceric acid dehydrogenase (PGAD) and serine-phosphate (serine-P) forming activities fluctuate with time after subculture and are higher in growing than confluent cells. This activity pattern was not common for other dehydrogenases in WIRL-3C cells, nor was it common for PGAD activity in other cultured cells. At time of subculture, cells are removed from spent medium, treated with trypsin, and fed fresh medium. None of these parameters causes the rise in activity; in contrast, reduction in cell density and the accompanying stimulation of growth do. PGAD activity decreases when growth is slowed either as the cells progress to the end of the culture cycle, when cells are treated with dexamethasone-phosphate (Dx-P) or dibutyryl cyclic AMP(cAMP) and theophylline or when the serum concentration of the medium is reduced to 0.2%. Under these conditions, decreased PGAD activity is paralleled by a decline in growth and DNA accumulation. PGAD activity in WIRL-3C cells is regulated in a manner closely resembling what has been observed previously in rat liver from the whole animal. The possible use of this system in studying regulation of gene expression in mammalian cells is discussed.

Animals↗

PubMed2025

Determinants of odorant receptor transcription and gene choice.

The mammalian olfactory system enables the detection of a wide variety of chemical compounds via the expression of a repertoire of olfactory receptors comprising the largest gene family in the mammalian genome. Olfactory sensory neurons (OSNs) each express only 1 odorant receptor (OR) gene. In mice, this requires activation of 1 OR gene and repression of over 1,400 other OR genes. In this review, we describe the mechanisms that support the transcription of OR genes and how these mechanisms impact which OR is expressed in each neuron. First, we discuss what is currently known about the role of transcription in OR choice. We then describe the role of specific features of OR genes and enhancers in the regulation of OR transcription. Finally, we discuss characteristics of OSNs which specify transcription of some OR genes while restricting the transcription of others.

Receptors, Odorant↗

PubMed2004

A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Aedes↗

PubMed2026

tRUBY: A convenient in planta tool for the detection of protein-DNA and protein-protein interactions.

Elucidating molecular interactions such as protein-DNA (PDIs) and protein-protein (PPIs) has traditionally relied on yeast-based 1-hybrid (1H) and 2-hybrid (2H) systems. To provide an alternative platform that better reflects the native cellular environment of plants, we optimized the tRUBY reporter system for 1H and 2H assays in Nicotiana benthamiana, enabling direct in planta analysis of PDIs and PPIs. Specifically, the 2A peptide sequence used for co-expressing the 3 betalain biosynthetic genes-responsible for the visible RUBY coloration-was replaced with T2A from the Thosea asigna virus in place of P2A or F2A from mammalian-pathogenic Picornaviridae viruses, improving biosafety for agricultural applications. The resulting tRUBY-1H and tRUBY-2H systems operate under near-physiological conditions with physiologically relevant expression levels, enabling quantitative, multiplexed, and directly compatible protein-level analyses, thereby offering high sensitivity and flexibility for advanced molecular studies. Ultimately, these systems demonstrate that the streamlined, cost-effective, and visually scorable in planta platform provided by RUBY is well-suited for intuitive, non-destructive monitoring of molecular interactions in plant tissues.

Nicotiana↗

PubMed1975

Use of mutant fibroblasts in the analysis of the regulation of cholesterol metabolism in human cells.

Analysis of mutant human fibroblasts deficient in a cell surface receptor for low density lipoproteins (LDL) has led to the delineation of an important, hitherto unrecognized, regulatory process for cholesterol metabolism. On normal cells, binding of LDL to this receptor regulates cholesterol metabolism by two mechanisms: (a) suppression of cholesterol synthesis and (b) facilitation of the rate of proteolytic degradation of the lipoprotein. In cells from homozygotes with the autosomal dominant disorder Familial Hypercholesterolemia, a nearly total reduction in LDL receptors results in two secondary abnormalities: (a) overproduction of cholesterol due to an inability of LDL to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, and (b) impairment in the rate of proteolytic degradation of LDL. Cells from heterozygotes possess about 50 per cent of the normal number of LDL recpetors; this leads to a concentration-dependent defect in regulation, so that attainment of rates of cholesterol synthesis and LDL degradation equal to that in normal cells requires a two to three-fold higher concentration of extracellular LDL in the heterozygote. The identification of this genetic regulatory defect in fibroblasts of heterozygotes with Familial Hypercholesterolemia makes available an in vitro system for studying the molecular mechanism by which a dominant mutation affects gene expression in mammalian cells.

Blood Proteins↗

PubMed1978

Type C RNA virus expression in systemic lupus erythematosus. New Zealand mouse model and human disease.

An antigen recognized by antisera produced against p30 (core) proteins of the four chief groups of mammalian type C viruses (murine, feline, RD-114 related to endogenous primate, and infectious primate group) is located in an immune-complex pattern in some renal glomeruli of human SLE patients with lupus proliferative glomerulonephritis but is not detected in normal or pathological control human kidneys. This antigen cross-reacts with p30 interspecies determinants shared by the four chief virus groups and cross-reacts with a partially purified antigen extracted from human SLE spleen. The human SLE spleen antigen cross-reacts with p30 group antigen of RD-114 virus but not of feline or murine viruses. Some host immunoglobulins eluted from a human SLE kidney by acid-buffer show antibody-like activity against p30 group antigen of RD-114 virus but not of simian, feline, or murine viruses.

Animals↗

PubMed2026

Bipolar disorder-associated variants in RAB5A disrupt its function in endolysosomal trafficking in fruit fly and mammalian systems.

Bipolar disorder is a severe neuropsychiatric disorder associated with significant morbidity and mortality. Although the heritability of bipolar disorder is among the highest of all neuropsychiatric disorders, the specific genetic etiologies for most affected individuals have remained elusive. This is due, in part, to difficulty confirming pathogenicity of genomic variants identified in bipolar disorder populations. From the Bipolar Exomes Browser (BipEX) project, we identified three missense variants in RAB5A in four individuals with bipolar disorder not found in controls. We used a Drosophila overexpression system to evaluate these bipolar disorder-associated variants in RAB5A. We determined that when bipolar disorder-associated RAB5A variants are expressed in fruit flies, synaptic neurotransmission, endolysosomal trafficking, viability, survival and wing morphology are all disrupted. We confirmed that certain bipolar disorder-associated RAB5A variants also disrupt early endosomal trafficking in mammalian cells. This confirms bipolar disorder-associated variants in RAB5A disrupt its normal function. Moreover, Drosophila melanogaster is a viable experimental system to test the pathogenicity of genomic variants associated with psychiatric disorders.

Journal Article↗

PubMed2026

Genome-wide characterization of the bZIP gene family in Rattus norvegicus and expression profiling analysis during brain development.

BACKGROUND: The brown rat (Rattus norvegicus) serves as a cornerstone model organism in biomedical research, particularly for understanding physiological homeostasis and stress responses. The basic leucine zipper (bZIP) transcription factor family is a pivotal regulatory network involved in growth, organogenesis, and neurodevelopment. Despite its importance, a systematic characterization of the bZIP gene family in rats has remained elusive. RESULTS: In this study, we performed a genome-wide identification of 61 RnbZIP genes, which were categorized into 10 distinct subfamilies based on phylogenetic relationships and chromosomal localization. Structural analysis revealed conserved motif arrangements within subfamilies, while collinearity analysis identified significant gene duplication events-predominantly tandem and segmental duplications-that have driven the evolutionary expansion of the RnbZIP family. Quantitative analysis showed that members within the same subfamily shared 45%-92% sequence similarity (calculated using the BLOSUM62 scoring matrix), and all duplicated gene pairs underwent strong purifying selection (Ka/Ks < 1). Comparative genomics across seven rodent species further underscored the evolutionary conservation and divergence of these factors. Expression profiling across diverse organs and brain developmental stages indicated that RnbZIP genes exhibit high tissue specificity. Notably, 10 candidate genes, including RnbZIP01, RnbZIP02, and RnbZIP08, demonstrated dynamic expression patterns during brain maturation, suggesting their essential roles in neurodevelopmental processes. CONCLUSIONS: Our findings provide a comprehensive structural and evolutionary framework for the RnbZIP gene family, highlighting their potential regulatory functions in rat organogenesis and brain development. This study establishes a valuable resource for further functional characterization of specific bZIP members in mammalian neurological systems.

Animals↗

PubMed2026

Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.

CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.

Humans↗

PubMed1979

Mutagenicity and cytotoxicity of nineteen heterocyclic mustards (ICR compounds) in cultured mammalian cells.

The mutagenicity and cytotoxicity of 19 ICR compounds, including 6 reported previously, have been determined in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyltransferase system. As with other physical and chemical agents, ICR 170 and 191 exhibit a phenotypic expression time of 7 to 9 days, independent of concentrations tested. Thirteen of these compounds are mutagenic. At equimolar concentrations, the compounds with the tertiary amine-type side chain (ICR 217, 340, 355, 368, 170, and 292) are more mutagenic than the compounds with the secondary amine-type side chain (ICR 449, 371, 191, and 372). All secondary amine types show a "plateau" in their concentration-dependent mutagenesis curves at 3 to 4 microM. Shortening of the side chain by one carbon (ICR 171) results in a reduced mutagenicity. Substitution of a sulfur atom for a nitrogen in the side chain (ICR 342) increases both mutagenicity and cytotoxicity. The presence of two 2-chloroethyl groups on the side chain (ICR 220) also results in greatly increased cytotoxicity and mutagenicity. When the 2-chloroethyl group of ICR 340, 372, 292, 191, or 170 is replaced by a 2-hydroxyethyl group (ICR 340-OH, 372-OH, 292-OH, 191-OH, or 170-OH), a mutagenically inactive compound results which remains toxic. Replacement of the amine linkage with an ether linkage (ICR 283) also yields a mutagenically inactive compound.

Animals↗

PubMed2026

Predictive design of tissue-specific mammalian enhancers that function in the mouse embryo.

Enhancers control tissue-specific gene expression across animals1. Although deep learning2,3 has enabled enhancer prediction and design in mammalian cell lines and non-mammalian model organisms4-10 (reviewed in a previous publication11), it remains unclear whether such approaches can operate within the regulatory complexity of mammalian genomes and tissues in vivo. Here we present a general strategy for designing tissue-specific enhancers that function reliably in mice. We use deep learning to train compact convolutional neural networks on curated chromatin accessibility data and fine-tune them by transfer learning on validated human and mouse enhancers. Guided by these models, we design 15 synthetic enhancers for the heart, limb and central nervous system in mouse embryos, all of which are active in their intended target tissue. These results demonstrate that mammalian enhancer function can be reliably inferred from DNA sequence alone, enabling the predictive de novo design of tissue-specific synthetic enhancers from modest training sets. This work establishes a generalizable framework for programmable control of mammalian gene expression in vivo, opening new avenues in functional genomics, synthetic biology and gene therapy.

Animals↗

PubMed1978

Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).

A monoclonal antibody derived by fusion of mouse myeloma cells with spleen cells from a mouse immunized with F9 teratocarcinoma cells is described. This antibody, which reacts with embryonal carcinoma cells of mouse and human origin and with some preimplantation stage mouse embryos, defines an embryonic stage-specific antigen. This stage-specific antigen (SSEA-1) is first detected on blastomeres of 8-cell stage embryos. Trophectodermal cells are transitorily positive; however, each cell in the inner cell mass eventually expresses this antigen.

Animals↗