The advancement of tissue clearing technology has significantly propelled neuroscience research. Nevertheless, the fluorescent proteins used in traditional transgenic mouse strains were not specifically optimized for tissue clearing procedures, resulting in a substantial decrease in fluorescent intensity after clearing. In this study, we developed the Ci1 reporter mouse strain (where Ci stands for the Chinese Institute for Brain Research, CIBR) based on the bright red fluorescent protein mScarlet. The Ci1 reporter exhibits no fluorescence leakage in various organs or tissue types and can be readily crossed with multiple tissue-specific Cre lines. Compared to the Ai14 mouse strain, the Ci1 reporter strain demonstrates lower non-specific leakage, stronger fluorescence intensity in different tissues, and better preservation of fluorescence following tissue clearing treatment. The creation of the Ci1 reporter provides a more effective tool for both neuroscience and other biomedical research applications.
Animals ; Luminescent Proteins/metabolism* ; Mice, Transgenic ; Mice ; Red Fluorescent Protein ; Brain/metabolism* ; Genes, Reporter ; Fluorescence

To develop a method for determining the antibody-dependent cell-mediated phagocytosis (ADCP) activity of human epidermal growth factor receptor 2 (HER2)-targeted antibody drug conjugate (ADC) based on the reporter gene assay, we established an ADCP activity assay with Jurkat/NFAT/FcγRIIa cells as the effector cells and BT474 as the target cells. Then, the target cell density, the ratio of effector to target cells, the target cell adhesion time, the incubation time for drug administration, and the induction time after adding effector cells were optimized by the method of design of experiment (DOE). The method showed a significant dose-response relationship, which was complied with the four-parameter equation: y=(A-D)/[1+(x/C)^B]+D. The durability ranges of the target cell density, the ratio of effector to target cells, the target cell adhesion time, the incubation time for drug administration, and the induction time after adding effector cells were (2.5-4.0)×10⁵ cells/mL, 3-5, 1.0-2.0 h, 0 h, and 5.0-6.0 h, respectively. The results of the methodological validation showed that the linear equation was y=1.106 8x-0.011 6, r=0.969 2. The established method showed the relative accuracy ranging from -6.59% to 2.98% and the geometric coefficient of variation less than 11% in the intermediate precision test. Furthermore, the method was target-specific. The method was then applied to the determination of ADCP activity of HER2-targeted ADC, demonstrating the result of (103.5±5.7)%. We developed a reporter gene assay for determining the ADCP activity of HER2-targeted ADC and the assay demonstrated high accuracy and good reproducibility, which proposes a highly efficient and approache for evaluating ADCP effect of this HER2-targeted ADC, and also provides a referable technique for characterizing the Fc effector functions of ADCs with diverse targets.
Humans ; Receptor, ErbB-2/immunology* ; Phagocytosis/drug effects* ; Immunoconjugates/immunology* ; Genes, Reporter ; Antibody-Dependent Cell Cytotoxicity ; Jurkat Cells

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.
Humans ; Mice ; Animals ; Transcription Factors/genetics* ; DNA-Binding Proteins/genetics* ; Receptors, Cytoplasmic and Nuclear/genetics* ; Genes, Reporter ; Luciferases/genetics*

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
Cloning, Molecular ; Escherichia coli/genetics* ; Genes, Reporter/genetics* ; Genetic Vectors/genetics* ; Lac Operon/genetics* ; Plasmids/genetics* ; beta-Galactosidase/genetics*

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.
Animals ; Animals, Genetically Modified ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins/genetics* ; Houseflies/genetics* ; Microinjections

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Alleles ; Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Circular/metabolism* ; Embryo, Nonmammalian ; Gene Editing/methods* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; Genotyping Techniques ; Green Fluorescent Proteins/metabolism* ; Integrases/metabolism* ; Luminescent Proteins/metabolism* ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/metabolism*

Animals ; Antigens, Surface/genetics* ; Cell Communication/genetics* ; Copulation/physiology* ; Fallopian Tubes/metabolism* ; Female ; Fertilization/genetics* ; GPI-Linked Proteins/genetics* ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/metabolism* ; Litter Size ; Luminescent Proteins/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism* ; Reproduction/genetics* ; Signal Transduction ; Sperm Count ; Sperm Motility/genetics* ; Spermatozoa/metabolism* ; Uterus/metabolism*

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

OBJECTIVE: To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region. METHODS: The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods. RESULTS: The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P＜0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3. CONCLUSION The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.
Adult ; Antigens, Neoplasm ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Cloning, Molecular ; Genes, Reporter ; HEK293 Cells ; Humans ; Leukemia, Monocytic, Acute ; genetics ; Luciferases ; Promoter Regions, Genetic