human ef1α promoter  (Addgene inc)

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: Stem Cell Reports

Article Title: Highly Efficient Genome Modification of Cultured Primordial Germ Cells with Lentiviral Vectors to Generate Transgenic Songbirds

doi: 10.1016/j.stemcr.2021.02.015

Figure Lengend Snippet: Cultured zfPGCs Were Efficiently Transduced with a Lentiviral Vector for eGFP and Colonized Host Embryo Gonads (A) The lentiviral vector rrl-hPGK-eGFP that was used to transduce zfPGCs in vitro and generate founder birds. (B–F) Microphotographs of cultured zfPGCs that were transduced with rrl-hPGK-eGFP for 48 h. Phase-contrast (B) and eGFP expression (C) of zfPGCs are shown at low magnification. Note that almost all zfPGCs were expressing the reporter gene. In (D–F), fluorescence images of a PGC cluster show the same cells expressing eGFP (D), being immunopositive for the PGC marker EMA-1 (E), and after DAPI staining (F). The inset in (E) shows a merged eGFP- and anti-EMA-1 image of a subset of cells (indicated by the arrow). Note that eGFP-expressing cells were EMA-1 immunopositive. (G and H) In microphotographs of cryosections produced from an adult founder ovary that showed eGFP expression and blue nuclear counterstain with DAPI are presented at low (G) and high (H) magnification. Note that in the ovary eGFP-positive germ cells of different sizes display various stages of differentiation. Scale bars represent 100 μm (C, G, and H) and 20 μm (D–F). (I–K) Photographs of agarose gels after electrophoretic separation and ethidium bromide staining of molecular weight markers (left lanes) and PCR products. PCR products that were obtained using the lentiviral vector plasmid (lane 1 in I and J) are shown as positive controls. Genomic DNA (gDNA) was isolated from a founder ovary (I, lane 2) and testis (I, lane 3), and from blood samples of three F1 birds, two males (J, lanes 2 and 3) and one female (J, lane 4). The right arrowhead in (I and J) points to the hPGK-promoter-eGFP amplicon (602 bp). To verify expression of the transgene by RT-PCR (K), RNA was isolated from the brain of a wild-type bird (K, lane 1) as well as brain (K, lanes 2 and 3) and liver (K, lanes 4 and 5) samples of different F1 birds. Although transgene expression could not be detected in skeletal muscle of transgenic birds by RT-PCR, the transgene was detectable in skeletal muscle gDNA of transgenic birds (K, lanes 7 and 8), in contrast to the wild type (K, lane 6). The right arrowhead in (K) points to the eGFP-WPRE amplicon (894 bp).

Article Snippet: The lentiviral vector for eGFP under control of the human EF1α promoter was purchased from SignaGen Laboratories (Gaithersburg, MD, USA).

Techniques: Cell Culture, Transduction, Plasmid Preparation, In Vitro, Expressing, Fluorescence, Marker, Staining, Produced, Molecular Weight, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay