Cag promoter expression This Also, its temperature sensitivity is high. Therefore, in lentiviral vectors With the CAG promoter, EGFP protein expression was 1. In contrast, the micro-promoters showed mRFP The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the In dramatic contrast to the commonly used universal CAG promoter, which drives EGFP expression in both GCL and inner nuclear layer (INL), the mouse and human Sncg demonstrated the best specificity among the promoters that we tested, as evidenced by restricted EGFP in GCL (Fig. The common eukaryotic promoters include elongation factor 1α (EF-1α) promoter; CAG promoter , which is composed of CMV immediate-early enhancer, chicken β-actin promoter , and the splicing acceptor of rabbit β-globin ; phosphoglycerate kinase (PGK) promoter ; ubiquitin c (UBC) promoter; and tissue-specific promoters. 12, 13 Thus, the constitutive CBh, CMV, and CAG promoters, which were delivered by parenchymal injection of AAV, worked specifically on neurons in the marmoset Despite the ability of the CAG promoter to drive gene expression in almost all tissues examined to date, its activity appears to be developmentally regulated within the T lymphocyte cell lineage. , 33 °C) increased the activity of the CMV and CAG promoters in HEK293F cells and increased recombinant protein productivity. 4-fold). (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. ( B ) Experimental procedure. However, such expression vectors sometimes fail to Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. However, the CAG promoter provided the highest levels of expression in all peripheral organs. In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosp Plasmid MXS_BidirectionalCAG from Dr. 101(1):16-22. Recent reports examined other common promoters, including SFFV, PGK, CMV7, and Ef1ɑ [29] , [30] for their transgene induction levels and pleiotropic effects on local chromatin and CAG promoter (pAeG) was 1. Fast accurate construct design for all The PGK promoter had predominant expression in both RGCs and AII amacrine cells. CAG is a synthetic hybrid promoter, consisting of promoter elements The EF-1a promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells Xiaoyin Wang a, b, #, Zhongjie Xu c, #, Zhengwei Tian a, #, Xi Zhang a, Danhua Xu a, Qin Li d, Junhe Zhang a, Tianyun Wang a, * a Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang, Henan, China b Henan d, Schematic showing SPH (single CAG), note that the expression of dCas9 and p65-HSF1 was driven by a single promoter. For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. CB7 is also a chicken β-actin promoter with 2. Please note: Addgene NGS is unable to fully resolve Abstract. Pierre Neveu's lab contains the insert Bidirectional CAG Promoter and is published in PLoS One. The reporter gene expression patterns presented here are achieved using the CAG promoter, which is known for its robust expression in multiple cell types. PGK promoter: Mouse: 500: Mouse phosphoglycerate kinase one promoter. 1985; Niwa et al. Connie Cepko. 0155177. promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. 1, 2 In the brain, distinct cell types are often defined by the expression of unique gene ensembles, emphasizing To determine whether AAV5 tropism differed between the MC and mPFC of Ai14 mice, AAV5 with Cre-recombinase expression driven by the ubiquitous CAG promoter was injected into the MC and mPFC of In fact, a recent survey showed that over 50% of rAAVs in 106 clinical settings used three universal promoters, the cytomegalovirus (CMV) promoter, which is the abbreviated term herein for the CMV enhancer and promoter, the chicken beta-actin (CBA) promoter, and the CAG promoter, which is a synthetic fusion of the CMV enhancer and the CBA IE enhancer/promoter/human β-globin intron) and CAG (human CMV IE enhancer/ chicken β-actin promoter/intron with rabbit β-globin 3’ splice site, also called the CBA or CAGGS promoter) (Boshart et al. 1P; signal-to-background ratio, 2:1). With the TBG410 promoter, no differences were observed between the Europe PMC is an archive of life sciences journal literature. 2008;9:2. [PMC free article Lower levels of expression in the brain were achieved using the CAG promoter, and lower still using the GAPDH promoter. 2010). GFP protein expression with the CAG promoter was highest at 3 weeks Some other constitutive promoters, including human β-actin (hACTB), human elongation factor-1α (hEF-1α), and cytomegalovirus early enhancer/chicken β-actin (CAG) promoters, have shown their merits in sustaining stable gene When the Rax and Hes1 expression vectors driven by the CAG promoter were coelectroporated with CAG-GFP into P0 rat retinas, most GFP-positive cells localized to the INL A promoter is a DNA sequence that can turn gene transcription on or off. (A): The top rows show the pAAVS1-CAG-EGFP construct, targeting site, and location of the 5 9 probe, When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. This difference may explain the weaker expression of the transgene under the control of the CAG promoter in transfected CHO cells. Our data . e. eCollection 2016. These results are consistent with a previous report which quantified Hybrid construct consisting of the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter. For this reason bicistronic vectors Finally, we tested if the Cre recombinase system, driven by the OXT promoter in one virus, can enhance the expression of a floxed-gene that is driven by the CAG promoter in another virus. To compare the strength of the CMV and CAG promoters, mRNA levels were measured by real-time PCR. 0x, 3. Interestingly, DNA sequence (466–526 nt) forms hairpin Yang et al. Tubulin was used as a control for Download CAG promoter. 4x, and >1000x the as the CAG promoter, which was 2 to 3-fold higher than the other two micro-promoters (Figure 3A). Consequently, other strong promoters including CAG (1733 bp), CBh (798 bp), and EF1α (1178 bp) are preferred for AAV in vivo use for broad expression. Results: In vitro transfection demonstrated a hierarchy of expression: hCMV-intron>CAG>EF1alpha>hCMV>>PGK. We generated AAV8 reporter vectors containing eGFP cDNA driven by the cytomegalovirus enhancer and chicken beta actin (CAG) hybrid promoter, GRK1 promoter, or human CAR promoter (Figure 1 A). Both the EF1α and the CAG promoter Similar results have been reported previously, which showed high neurotropic transgene expression following direct injection of AAV8 or AAV9 with the CAG promoter in the marmoset brain. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. The expression levels and stability of the transgenes were To explore how promoter methylation controls the exogenous gene expression in details, methylation statuses of full-length CAG promoter regions in two types (EGFP positive In the present study, the transduction efficiency in the liver following intraperitoneal (IP) and intravenous (IV) injections of rAAV8 with the CAG, TBG669 and TBG410 promoters was We offer a variety of promoters to drive target gene expression including promoters for mammalian, plant, zebrafish, yeast and drosophila gene expression. In vivo luminometry demonstrated that the CAG construct produced 2. The Chicken beta actin contains a CpG island that can help to keep the promoter active for longer in stable culture when compared to The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian expression vectors. Information Expression Level Unknown Cloning Method Unknown Size 584 Notes Hybrid construct consisting of the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter. 108:193 The SV40 promoter has been shown to drive relatively low expression in HEK293 cells [18,48,49], but whether the CMV or CAG promoter drives stronger transgene expression appears to be more context With the CAG promoter, EGFP protein expression was 1. In efforts to generate a constitutive CRISPRa pluripotent stem cell line, the AAVS1 safe harbor locus was targeted with dCas9-VPR driven by the CAG promoter in WTC11 hiPSCs (Figs. Stable Unspecified Constitutive Unspecified the CAG promoter drove gene expression in the ES cells more efficiently than the (strongly silenced) CMV promoter, whereas the EF1a and the phosphoglycerate kinase (PGK) promoters showed much stronger transgene expression and were thereby claimed to be the promoters of choice for HuES cells. Targeting the AAVS1 safe harbor locus for constitutive CRISPRa expression. 5‑fold higher with the use of IV injection than with IP injection. All founders were first assayed for baseline, non The comparison of the bidirectional promoter variants relying on two different core promoters (variants ①–④) revealed that a CAG-type promoter (variant ①) supported high detector expression that was not matched by the Vectors containing the CAG promoter offer a valuable tool for the long term expression of transgenes during stem cell differentiation towards mesoderm, while the CMV and beta-actin promoters lead to very poor transgene expression during this process. e , Histogram of FACS analysis of a SPH (single CAG) -OminiCMV-Ents- Actb colony. But its temperature sensitivity is low. 027; Fig. doi: 10. Isolated RBPMS-positive cells in INL that co-localize with CAG promoter, cell cycle speed, cell size, MKL1/SRF signaling, Yamanaka reprogramming 1 | INTRODUCTION The somatic cells amenable to switching into pluripotency upon expression of the Yamanaka factors are considered to exist stochasti-cally. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant Alternatively, a variety of strong promoters, including human elongation factor-1 alpha (EF-1α) promoter and CAG promoter (a combination of the CMV immediate early enhancer and the chicken β-actin promoter), have GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. The CAG promoter has been used to deliver Cre recombinase and various regeneration-associated genes in the The CAG promoter provides long term expression of transgenes in these cells however the system is most effective when the cells can be put under a selective pressure to express the transgene. pCAGGS-GFP has CAG promoter containing a CMV immediate early enhancer and a chicken β-actin promoter, which is a strong promoter driving the eukaryotic gene expression; pEGFP-N1 has CMV promoter which is the most widely used promoter in many commercial and customized Compared with the CMV promoter, the enhancement was the highest for CHEF1-α, which improved transgenic eGFP expression by 2. Matsuda et al Proc Natl Acad Sci U S A. Comparison of promoters for gene expression in DRG. , 1991). SnapGene is the easiest way to plan, visualize and Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. 9-fold, followed by HEF1-α (2. Orientation of the 3′-CMV promoter-independent gene expression. Explore Over 2. The AAV-ChAT transgene expression cassette should be a useful tool for the study of cholinergic neurons in mice, and the broader transduction area of AAV-S warrants The cytomegalovirus promoter CMV is one of the most commonly used and also the strongest constitutive promoters. A plasmid for a minimal human synapsin promoter (485 bp) driving expression of yellow The chicken b actin (CAG) promoter drove EGFP expression at the AAVS1 locus. The composite CAG promoter was chosen because it functions efficiently in murine embryonic stem cells and in transgenic mice . In conclusion, our data show that the CAG promoter is a strong promoter for recombinant Unlike the more heterogeneous activation observed with the EF1α, CBh, and CMV promoters, the CAG promoter induced distinctly uniform and potent gene expression for each of the tested cell lines CAG promoter drives ubiquitous gene expression in a variety of tissues, including hematopoietic CD45+ cells, which are comprised of lymphoid and myeloid lineages. 6x, 3. 3a, b). 2016 May 6;11(5):e0155177. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate‐early enhancer combined with the chicken beta‐actin promoter (CAG) promoter, as compared Because of this robust activity, the CAG promoter is quite popular and has been shown to provide durable expression in viral vectors such as AAV [20,22]. BMC Cell Biol. EGFP construct was microinjected in fertilized SD oocytes, resulting in 12 Cre reporter founder rats. CAG启动子. To compare the performance of the CMV and CAG promoter in DRG neurons, titre-matched single-stranded AAV5 vectors expressing eGFP (Figures 1a promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. This constitutive promoter is widely recognized as one of the strongest expression drivers alongside CAG promoter (Qin et al. The CMV immediate enhancer/β-actin The compound promoter, CAG, was found to yield the highest levels of expression at approximately 9–10 fold the level of the endogenous ROSA26 promoter. Gene. 7k Plasmids: Basic Cloning Vectors | More Plasmid Sets. When the RFP expression driven the six promoters in the stable 293 lines was compared, we found that the CMV and CMV-IVS promoters exhibited the highest RFP expression, while the RFP expression driven by hEFH, CAG-hEFH, and SV40 Plasmid: CAG promoter. Surprisingly, GFP mRNA levels continued to The EF1α promoter drove the highest level of GFP expression, followed by the CAG promoter, the PGK promoter, the GALV LTR, and the MSCV LTR (Supplementary Figure S2b and c). According to tests on cell cultures, it provided a high constant expression in various cell CAG promoter was constructed in the lab of Dr Jun-ichi Miyazaki [3] [4] from the following sequences: (C) the cytomegalovirus (CMV) early enhancer element,(A) the promoter, the first exon and the first intron of chicken beta-actin gene,(G) the splice acceptor of the rabbit beta-globin geneThe resulting synthetic element was used in the pCAGGS expression vector. In comparison, the expression of the CAG promoter is high. a Schematic of the CRISPR-Cas12a systems driven by different promoters (CAG, EF1a Core, CMV, and Interestingly, the EF1 promoter exhibited the highest activation compared to the human cytomegalovirus (CMV) or CMV-enhanced chicken beta-actin (CAG) promoters as determined by flow cytometry and Promoter Expression Level: The plasmid vector contains the mammalian CAG promoter which is a synthetic composite of the CMV immediate early enhancer followed by the CBA promoter and the rabbit beta globin intron. SnapGene is the easiest way to plan, visualize and document your everyday molecular biology procedures. 1-3 Within such a model, the rare cells known to have high repro- Specificity of transgene expression of ChAT neurons over other cells was 7-fold higher, and efficiency was 3-fold higher for the ChAT promoter compared with the CAG promoter. As mentioned above, promoter activity is affected by transcription factor In fact, a recent survey showed that over 50% of rAAVs in 106 clinical settings used three universal promoters, the cytomegalovirus (CMV) promoter, which is the abbreviated The term CBA promoter is synonymous with the CAG promoter (Niwa et al. 2004 Jan 6. 人们认识到CMV启动子的不足后，通过将不同物种的启动子“复制粘贴”到一起，构建了一系列人工启动子，比如所CAG、CBA等等启动子。CAG启动子包含CMV病毒启动子的增强子序列，鸡β-actin的启动子以及兔β-Globin Through analyzing the correlation between the methylation level of CAG promoter and the expression level of DNMT, we have also found that the methylation of CAG promoter is associated with the expression of Dnmt3b. However, mouse CMV and CAG Rhodopsin K and Cone Arrestin Promoters Display Robust Activity in Photoreceptors but Poor Transgene Expression in Cell-Based Systems. Click to refer colleagues and get 30% off CAG: CMV early enhancer fused to modified chicken β-actin promoter: Strong promoter. Consequently, Dnmt3b may stimulate the methylation of CAG promoter so as to inhibit the expression of transgene. When HS4 was juxtaposed to the 5′ and 3′ regions of the transgene cassette, the difference Upon Cre-mediated recombination, the lacZ/STOP cassette is deleted, which activates CAG-promoter controlled expression of EGFP. MFI was analyzed by flow To optimize scFv expression in transgenic cells, we compared the transcriptional efficiency between CAG promoter and EF1α promoter and found that both of the mRNA and protein levels of the scFv When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. The significant expression in the peripheral organs suggested escape of the vector from the CSF in systemic circulation. 1186/1471-2121-9-2. All three promoters demonstrated weak fluorescence 24 hr after infection (Fig. To ensure the ubiquitous expression of the human ACE2 gene we used the human EF1a promoter. In lentiviral vectors, the CAG promoter poses major limitations, likely due to its high GC content, which can lead to low viral titers during virus packaging. However, its expression profile in the The main difference between CMV and CAG promoter is that the transgene expression of the CMV promoter is low, whereas the transgene expression of the CAG promoter Vectors containing the CAG promoter can be used to express transgenes in murine CCE cells and during differentiation into mesoderm. 1 D–F. Subretinal Mammalian expression vector for expression of GFP (CAG promoter) Depositing Lab. Then, we found that a mild low temperature (i. The structure difference among the three plasmids was shown in Fig. The time course of mRFP expres-sion showed that the transcriptional activity from the CAG promoter was surprisingly rapid in these cells, as mRFP expression was visible within 24 h after transduction. To avoid the high variation in Finally, we tested if the Cre recombinase system, driven by the OXT promoter in one virus, can enhance the expression of a floxed-gene that is driven by the CAG promoter in another virus. 1371/journal. To create transgenic rat Cre reporter lines, the CAG-loxP. Nevertheless, it is still unsettled which promoter is to When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3 x 10(7) genome copies, and continued to increase in a dose- dependent manner. Download Plasmid Open in SnapGene. 1991), are useful for robust and long-term transgene expression in a broad range of cell types in preclini - cal animal CMV and CAG promoter transcription activities differ in chicken embryonic fibroblasts. dna file. 1 B,C). Publication. Using pronuclear microinjection of the transgene construct, we obtained six founders with the insertion of the EF1a-hACE2 transgene, from which four independent mouse This reporter is designed around a bidirectional CAG promoter driving the expression of two genes encoding for two fluorescent proteins: one gene contained in its 3'-UTR a binding site for the Rat hippocampal organotypic slices were infected with AAV9 vectors using a CMV, CBA, or CBh promoter driving the expression of GFP. a Orientation of the 3′-CMV promoter in the CMV-KLF16-CMV construct was flipped, termed CMV-KLF16-CMV (flipped), and then expression of KLF16 protein was assessed by Western blot analysis after transfecting the plasmid including indicated comparative vectors in HEK293. In particular, CD4 and CD8-expressing, thymic immature T cells displayed lower levels of the GFP marker when compared with both bone marrow precursors In this work, we set out to create mice susceptible to the SARS-CoV-2 coronavirus. To determine vector copy numbers in cells transduced with each of the vectors, we isolated genomic DNA from undifferentiated mES cells and then performed quantitative PCR pPB-CAG-EGFP drives EGFP expression under the CAG promoter. , 2005). CAG promoter (AAV1 and AAV9 serotypes) We began our analysis by testing the CAG promoter. pCAG-PBase has a piggyBac transposase, which recognizes the terminal region (TR) in piggyBac donor plasmids. When eGFP was replaced with hepatitis B surface antigen, the CAG pro-moter still showed the highest transgene expression. The Chicken beta actin contains a CpG island that can help to keep the promoter active for longer in stable culture when compared to As some artificial promoters can be prone to strong transgene silencing, especially in pluripotent stem cells (PSC), we employed the CMV i/e enhancer chicken β-actin rabbit globin-intron (CAG The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver. 2010; Norrman et al. 6-fold higher than that of the CMV promoter (pCeG) (P = 0. gene expression due to a high copy number of plasmid per cell, certain experiments may require lower levels of over-expression that may be more physiologically relevant. demonstrated that the CAG promoter can drive transgene expression in chick embryo cells 33, whereas we transfected CHO cells. CAG In these processes, constitutive promoters are useful tools due to their high level of expression in most cell types. With the TBG410 promoter, no differences were The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. After selection, we identified several clones that correctly integrated the transgene at the Strong expression promoter; highly efficient in stem cells. pone. 1a, b, S1a). Promoter efficacy was monitored by evaluating GFP production daily over a 5-day time course. SnapGene. Promoter Expression Level: The plasmid vector contains the mammalian CAG promoter which is a synthetic composite of the CMV immediate early enhancer followed by the CBA promoter and the rabbit beta globin intron. Importantly, the CAG promoter CAG promoter can drive transgene expression in the cochlea cells at a highly functional level (Liu et al. The constitutive cytomegalovirus (CMV) enhancer/chicken β-actin promoter (CAG) promoter was recently used On the basis of correlation analysis of the methylation status of CAG promoter and the expression level of EGFP, we found that the methylation levels of three CpG loci (101, 108 and 115 nt) are negative correlation with exogenous gene expression quantity (Figs Figs6B 6B and and5B 5B). To avoid the high variation in In conclusion, we first identified the impact of the CAG promoter on the expression of recombinant proteins as both transient and stable transgenes in HEK293F cells. Here, we compare the promoter activity of the CMV, chicken β-actin and CAG promoters in undifferentiated murine embryonic CCE cells as well as during differentia-tion to mesoderm. In the present study, we systematically eva- In fact, a recent survey showed that over 50% of rAAVs in 106 clinical settings used three universal promoters, the cytomegalovirus (CMV) promoter, which is the abbreviated term herein for the CMV enhancer and promoter, the chicken beta-actin (CBA) promoter, and the CAG promoter, which is a synthetic fusion of the CMV enhancer and the CBA Comparison of the expression level and the activity of the CRISPR-Cas12a systems using different promoters.
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