A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT

T cell immunoglobulin and ITIM domain(TIGIT)is a novel immune checkpoint that has been considered as a target in cancer immunotherapy.Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable.Here,we designed a reporter gene assay comprising two cell lines,namely,CHO-CD112-CD3 scFv,which stably expresses CD 112(PVRL2,nectin-2)and a membranebound anti-CD3 single-chain fragment variable(scFv)as the target cell,and Jurkat-NFAT-TIGIT,which stably expresses TIGIT as well as the nuclear factor of activated T-cells(NFAT)response element-controlled luciferase gene,as the effector cell.The anti-CD3 scFv situated on the target cells activates Jurkat-NFATTIGIT cells through binding and crosslinking CD3 molecules of the effector cell,whereas interactions between CD 112 and TIGIT prevent activation.The presence of anti-TIGIT mAbs disrupts their interaction,which in turn reverses the inactivation and luciferase expression.Optimization and validation studies have demonstrated that this assay is superior in terms of specificity,accuracy,linearity,and precision.In summary,this reliable and effective reporter gene assay may potentially be utilized in lot release control,stability assays,screening,and development of novel TIGIT-targeted therapeutic antibodies.

MEDICINAL PLANTS OF THE GENUS LEONURUS AND SEVENTEEN OF THEIR ISOLATED CONSTITUENTS SCREENED FOR EFFECTS ON PPARa,β/δ, and γ IN AN IN VITRO LUCIFERASE REPORTER GENE ASSAY

Leonurus japonicus Houtt.is used in TCM to treat the metabolic syndrome.However,up to now,no active constituents could be identified.Here we describe the isolation of 17 dominant constituents of L.japonicus and the related European herb Leonurus cardiaca L.-namely7R-chloro-6-desoxy-harpagide,ajugol,campneoside II,chicoric acid,ferulic acid,harpagide,isoacteoside,

Effects of CMV Enhancer on Activity and Specificity of Bovine MyoG Gene Promoter

Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.

Simple and rapid determination of dioxin in fish and sea food using a highly sensitive reporter cell line,CBG 2.8D

Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bioassay method is needed for determination of dioxins in food samples.In this study,we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples.The samples were extracted by shaking with water/isopropanol(1:1 v/v)and hexane and cleaned-up by a multi layered silica gel column and an alumina column,then analyzed using CBG 2.8 D cell line.We compared the results obtained from the CBG 2.8 D cell assay to those obtained from conventional High-Resolution Gas Chromatography-High Resolution Mass Spectrometry(HRGC-HRMS)analysis.Good correlations were observed between these two methods(r^2=0.93).While the slope of regression line was 1.76,the bioanalytical equivalent(BEQ)values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568(the reciprocal of 1.76).In conclusion,CBG 2.8 D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.

Polymorphisms and functions of the aldose reductase gene 5' regulatory region in Chinese patients with type 2 diabetes mellitus

Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.