Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Gruh, Ina; Wunderlich, Stephanie; Winkler, M; Schwanke, Kristin; Heinke, Jennifer; Blömer, Ulrike; Ruhparwar, Arjang; Rohde, B; Li, Ren‐Ke; Haverich, Axel; Martin, Ulrich

doi:10.1002/jgm.1122

Cited by 49 publications

(31 citation statements)

References 33 publications

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“…Lentiviral expression constructs containing cDNA encoding human CYP2J2 were generated. This transgene cassette employs the cytomegalovirus (CMV) immediate-early promoter (20). The lentiviral vector containing the puromycin resistance gene (CMV-eGFP-IRES-Puro plasmid) and the packaging vectors (VSV-G expressing envelop plasmid and another plasmid containing gag, pol and rev genes) were kindly provided by Dr. Yibing Qyang (Yale Cardiovascular Research Center, Yale School of Medicine, USA).…”

Section: Reagentsmentioning

confidence: 99%

Role of cytochrome P450 2J2 on cell proliferation and resistance to an anticancer agent in hepatocellular carcinoma HepG2 cells

et al. 2017

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Abstract. The present study examined the role of human cytochrome P450 2J2 (CYP2J2) on cell proliferation and resistance to an anticancer agent using stable hepatocellular carcinoma HepG2 cells overexpressing CYP2J2. Overexpression of CYP2J2 significantly increased HepG2 cell proliferation and the expression levels of cell cycle regulatory proteins, including cyclin D1, cyclin E, cyclin-dependent kinase (Cdk)2 and Cdk4. CYP2J2-overexpressing HepG2 cells exhibited high levels of Akt phosphorylation compared with those observed in wild-type HepG2 cells. Although Akt phosphorylation in both cell lines was significantly attenuated by LY294002, a specific phosphoinositide 3-kinase/Akt signaling inhibitor, the levels of Akt phosphorylation following treatment with LY294002 were higher in CYP2J2-overexpressing HepG2 cells than in wild-type HepG2 cells. Cell counting revealed that proliferation was reduced by LY294002 in both cell lines; however, CYP2J2-overexpressing HepG2 cell numbers were higher than those of wild-type HepG2 cells following treatment with LY294002. These results indicated that increased cell proliferation by CYP2J2 overexpression is mediated by increased Akt activity. It was also demonstrated that doxorubicin, an anticancer agent, reduced cell viability, induced a significant increase in the B-cell lymphoma (Bcl)-2 associated X protein (Bax)/Bcl-2 ratio and decreased pro-caspase-3 levels in wild-type HepG2 cells. However, the doxorubicin-induced reduction in cell viability was significantly attenuated by enhanced upregulation of CYP2J2 expression. The increase in the Bax/Bcl-2 ratio and the decrease in pro-caspase-3 levels were also recovered by CYP2J2 overexpression. In conclusion, CYP2J2 serves important roles in cancer cell proliferation and resistance to the anticancer agent doxorubicin in HepG2 cells.

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Section: Reagentsmentioning

confidence: 99%

Role of cytochrome P450 2J2 on cell proliferation and resistance to an anticancer agent in hepatocellular carcinoma HepG2 cells

et al. 2017

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“…63,64 Adding the CMV enhancer sequence (enhSyn1) additionally improves the neuron-specific transcriptional activity of the hSyn1 promoter, in line with previous reports. 67,68 Another surprising finding was the different transduction patterns induced by two highly similar viral vector constructs consisting of different lengths (364 bp and 1291 bp) of the CaMKIIα promoter. We found that in V1, the short 0.4 kb CaMKIIα-promoter transduces neurons in all layers, with the least efficiency in layers IVb and IVcα.…”

Section: Promoter-dependent Transgene Expression Levelsmentioning

confidence: 99%

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

et al. 2015

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Abstract. Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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“…Since GATA-4 and GATA-6 function in transcription through a GAT(A/T) motif [17] [18], it seems likely that the CMV enhancer with GATA motifs (Figure 4(b)) immediately responds to pre-existing GATA-6 in P19CL6 cells on DMSO treatment. Actually, the CMV enhancer induces efficient cell-type specific expression of genes such as those of atrial natriuretic factor and MLC-2v in cardiomyocyte cell line HL-1 [19], which expresses GATA-4 and GATA-6 [20]. Consistent with this finding, the SV40 enhancer composed of the 72 bp tandem repeat [21] without GATA motifs (NCBI Reference Sequence NC_001669) abolished the cardiac specificity [9].…”

Section: Discussionmentioning

confidence: 68%

The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2v Promoter during P19CL6 Cell Differentiation

Wakayama¹,

Ohashi²,

Fujimoto³

et al. 2017

AJMB

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The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2v pro /EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.

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Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Cited by 49 publications

References 33 publications

Role of cytochrome P450 2J2 on cell proliferation and resistance to an anticancer agent in hepatocellular carcinoma HepG2 cells

Role of cytochrome P450 2J2 on cell proliferation and resistance to an anticancer agent in hepatocellular carcinoma HepG2 cells

Serotype-dependent transduction efficiencies of recombinant adeno-associated viral vectors in monkey neocortex

The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2v Promoter during P19CL6 Cell Differentiation

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