Journal: Scientific Reports

Article Title: Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells

doi: 10.1038/s41598-022-09887-2

Figure Lengend Snippet: Experimental workflow of the study, promoter characteristics and vector designs. ( A , B ) Schematic representation of the workflow: Promoter strengths in the transient expression context were quantified using dual luciferase assay and flow cytometry analysis. ( A ) All nine promoters were compared based on luciferase activity in CHO cell variants and HEK-293T cells (Experimental setting 1). To corroborate the dual luciferase reporter findings, one weak and three relatively strong promoters were cloned into the dual fluorescence reporter system and further investigated by flow cytometry analysis (Experimental setting 2). ( B ) The activity of five relatively strong promoters selected and prioritized from ( A ) was evaluated in stable transfectants of CHO-DG44 suspension cells by dual luciferase assay. Further experiments were performed to analyze the expression of Fluc and Rluc genes, as well as the copy number of Rluc and DHFR genes. ( C ) The list of well-known constitutive promoters tested herein. Color codes represent the origin of promoters. ( D ) The table depicts promoter lengths. ( E ) Representative vector maps of all-in-one reporter systems. In the dual luciferase reporter system, the variable promoter (CMV-mIE) was replaced by SV40, HSV-TK, EF1α, EFS, UBC, PGK, CHEF1α and CAG promoters. In the dual fluorescent reporter system, the variable CMV-mIE was replaced by SV40, HSV-TK and CHEF1α promoters. For stable expression analysis, the constructs with CMV-mIE, SV40, UBC, CHEF1α and CAG promoters were engineered to coexpress Rluc and DHFR genes separated by an IRES sequence, creating a bicistronic expression cassette. The schematics in ( E ) are created with BioRender.com.

Article Snippet: Accordingly, the variable CMV-mIE promoter was successfully replaced by the following promoters: simian virus 40 (SV40) enhancer/early promoter (template: pcDNA3.1( +)/myc-His A, Invitrogen), herpes simplex virus thymidine kinase (HSV-TK) promoter (template: pRL-TK, Promega), mouse phosphoglycerate kinase 1 (PGK) promoter (template: TTI-GFP, Scott Lowe laboratory), human eukaryotic translation elongation factor 1 alpha (EF1α) promoter (template: pENTR5’/EF1αp, Invitrogen), human EF1α short (EFS) promoter (template: lentiCRISPR v2, Addgene plasmid #52961), Chinese hamster EF1α (CHEF1α) promoter (template: CHO-WT gDNA, GenBank accession number AY188393.1, position 11151–12623 or − 463 to + 1010 according to transcription start site), cytomegalovirus (CMV) early enhancer element fused to the chicken beta-actin (CAG) promoter (template: pCAG-ERT2CreERT2, Addgene plasmid #13777), and human ubiquitin C (UBC) promoter (template: pLV hUbC-VP64 dCas9 VP64-T2A-GFP, Addgene plasmid #59791).

Techniques: Plasmid Preparation, Expressing, Luciferase, Flow Cytometry, Activity Assay, Clone Assay, Fluorescence, Suspension, Construct, Sequencing

Journal: Scientific Reports

Article Title: Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells

doi: 10.1038/s41598-022-09887-2

Figure Lengend Snippet: Experimental workflow of the study, promoter characteristics and vector designs. ( A , B ) Schematic representation of the workflow: Promoter strengths in the transient expression context were quantified using dual luciferase assay and flow cytometry analysis. ( A ) All nine promoters were compared based on luciferase activity in CHO cell variants and HEK-293T cells (Experimental setting 1). To corroborate the dual luciferase reporter findings, one weak and three relatively strong promoters were cloned into the dual fluorescence reporter system and further investigated by flow cytometry analysis (Experimental setting 2). ( B ) The activity of five relatively strong promoters selected and prioritized from ( A ) was evaluated in stable transfectants of CHO-DG44 suspension cells by dual luciferase assay. Further experiments were performed to analyze the expression of Fluc and Rluc genes, as well as the copy number of Rluc and DHFR genes. ( C ) The list of well-known constitutive promoters tested herein. Color codes represent the origin of promoters. ( D ) The table depicts promoter lengths. ( E ) Representative vector maps of all-in-one reporter systems. In the dual luciferase reporter system, the variable promoter (CMV-mIE) was replaced by SV40, HSV-TK, EF1α, EFS, UBC, PGK, CHEF1α and CAG promoters. In the dual fluorescent reporter system, the variable CMV-mIE was replaced by SV40, HSV-TK and CHEF1α promoters. For stable expression analysis, the constructs with CMV-mIE, SV40, UBC, CHEF1α and CAG promoters were engineered to coexpress Rluc and DHFR genes separated by an IRES sequence, creating a bicistronic expression cassette. The schematics in ( E ) are created with BioRender.com.

Article Snippet: Accordingly, the variable CMV-mIE promoter was successfully replaced by the following promoters: simian virus 40 (SV40) enhancer/early promoter (template: pcDNA3.1( +)/myc-His A, Invitrogen), herpes simplex virus thymidine kinase (HSV-TK) promoter (template: pRL-TK, Promega), mouse phosphoglycerate kinase 1 (PGK) promoter (template: TTI-GFP, Scott Lowe laboratory), human eukaryotic translation elongation factor 1 alpha (EF1α) promoter (template: pENTR5’/EF1αp, Invitrogen), human EF1α short (EFS) promoter (template: lentiCRISPR v2, Addgene plasmid #52961), Chinese hamster EF1α (CHEF1α) promoter (template: CHO-WT gDNA, GenBank accession number AY188393.1, position 11151–12623 or − 463 to + 1010 according to transcription start site), cytomegalovirus (CMV) early enhancer element fused to the chicken beta-actin (CAG) promoter (template: pCAG-ERT2CreERT2, Addgene plasmid #13777), and human ubiquitin C (UBC) promoter (template: pLV hUbC-VP64 dCas9 VP64-T2A-GFP, Addgene plasmid #59791).

Techniques: Plasmid Preparation, Expressing, Luciferase, Flow Cytometry, Activity Assay, Clone Assay, Fluorescence, Suspension, Construct, Sequencing

Journal: Scientific Reports

Article Title: Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells

doi: 10.1038/s41598-022-09887-2

Figure Lengend Snippet: Cross validation of bioluminescence based results and analysis of promoter strength using the dual fluorescence reporter system in transient expression settings. Promoter strength was investigated by flow cytometry to verify bioluminescence based analyses while also demonstrating that the reporter system can function with fluorescence signals. The vector panel carrying CMV-mIE, SV40, HSV-TK and CHEF1α promoters was transiently transfected into CHO cell variants. At 48 h post-transfection, flow cytometry was conducted to evaluate promoter activities on ( A ) CHO-WT, ( B ) CHO-DG44 adherent and ( C ) CHO-DG44 suspension cells. ( D ) Similar to the dual luciferase reporter system, the ratio of eGFP/tdTomato fluorescence signals provides a systematic and quantitative comparison of the promoters. The strength of each promoter was expressed as the ratio of eGFP positive cells (Q1 + Q2) divided by the percentage of tdTomato positive cells (Q2 + Q3).

Article Snippet: Accordingly, the variable CMV-mIE promoter was successfully replaced by the following promoters: simian virus 40 (SV40) enhancer/early promoter (template: pcDNA3.1( +)/myc-His A, Invitrogen), herpes simplex virus thymidine kinase (HSV-TK) promoter (template: pRL-TK, Promega), mouse phosphoglycerate kinase 1 (PGK) promoter (template: TTI-GFP, Scott Lowe laboratory), human eukaryotic translation elongation factor 1 alpha (EF1α) promoter (template: pENTR5’/EF1αp, Invitrogen), human EF1α short (EFS) promoter (template: lentiCRISPR v2, Addgene plasmid #52961), Chinese hamster EF1α (CHEF1α) promoter (template: CHO-WT gDNA, GenBank accession number AY188393.1, position 11151–12623 or − 463 to + 1010 according to transcription start site), cytomegalovirus (CMV) early enhancer element fused to the chicken beta-actin (CAG) promoter (template: pCAG-ERT2CreERT2, Addgene plasmid #13777), and human ubiquitin C (UBC) promoter (template: pLV hUbC-VP64 dCas9 VP64-T2A-GFP, Addgene plasmid #59791).

Techniques: Biomarker Discovery, Fluorescence, Expressing, Flow Cytometry, Plasmid Preparation, Transfection, Suspension, Luciferase, Comparison

Journal: Scientific Reports

Article Title: Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells

doi: 10.1038/s41598-022-09887-2

Figure Lengend Snippet: Validation of the vector toolkit in stable transfectants of CHO-DG44 cells. ( A ) Luciferase activity in stable CHO-DG44 pools electroporated with the select panel of vectors (UBC, SV40, CMV-mIE, CAG, CHEF1α and NP). Luciferase assay was carried out in three consecutive passages (Passage 10 to 12), and each promoter activity was concurrently assessed. Quantification of promoter strengths were carried out by dividing the Fluc signal to the Rluc signal. ( B ) mRNA expression of Fluc and Rluc genes was verified by qRT-PCR. Much like the luciferase assay results, CHEF1α yielded the highest transcriptional activity in the stable transfection setting, while CMV-mIE displayed the lowest promoter efficacy. ( C ) Relative DHFR and Rluc copy numbers were calculated according to the internal control gene, GAPDH. Bright Green Master Mix was used for both qRT-PCR assays ( B ) and transgene copy number analysis by qPCR ( C ).

Article Snippet: Accordingly, the variable CMV-mIE promoter was successfully replaced by the following promoters: simian virus 40 (SV40) enhancer/early promoter (template: pcDNA3.1( +)/myc-His A, Invitrogen), herpes simplex virus thymidine kinase (HSV-TK) promoter (template: pRL-TK, Promega), mouse phosphoglycerate kinase 1 (PGK) promoter (template: TTI-GFP, Scott Lowe laboratory), human eukaryotic translation elongation factor 1 alpha (EF1α) promoter (template: pENTR5’/EF1αp, Invitrogen), human EF1α short (EFS) promoter (template: lentiCRISPR v2, Addgene plasmid #52961), Chinese hamster EF1α (CHEF1α) promoter (template: CHO-WT gDNA, GenBank accession number AY188393.1, position 11151–12623 or − 463 to + 1010 according to transcription start site), cytomegalovirus (CMV) early enhancer element fused to the chicken beta-actin (CAG) promoter (template: pCAG-ERT2CreERT2, Addgene plasmid #13777), and human ubiquitin C (UBC) promoter (template: pLV hUbC-VP64 dCas9 VP64-T2A-GFP, Addgene plasmid #59791).

Techniques: Biomarker Discovery, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Stable Transfection, Control