Part:BBa_K5036021

UAS Trans CMV enhancer

Part Description

it is a a Powerful Tool for Gene Expression which is derived from the cytomegalovirus (CMV). it can significantly boost gene expression in various cell types. it works by binding to specific transcription factors, which then recruit other proteins to the promoter region of a gene. This complex interaction helps to initiate and sustain gene transcription.

Usage

This part is fused with GAL4, dcas9(c), VP64. it acts as a powerful transcription activator where it induce transcription and increased gene expression so after receptor activation, it will be directed with dCas9,VP64 and GAL4 to increases expression of YAP-1.

this figure illustrates the structure of UAS TransCMV enhancer .

Characterization by Mathematical Modeling

The model provides the activation kinetics of CMV trans-enhancer which occurs subsequent to upon releasing of the d-Cas9 system to initiate the transcription of the YAP-1.The result shows increase in the transcription level of YAP-1 which implies successful CMV trans-enhancer activation based on parametric values from literature

Graph(1). Illustrates the relation between activation level of CMV trans-enhancer (Blue line) for increasing the transcription level of YAP-1 (Black line) .

Experimental Characterization

dCas9-VP64-GAL4 expression vector and a UAS-CMV trans-enhancer were constructed to investigate the effectiveness of the GAL4-UAS system for CRISPR-assisted trans-enhancer activation . Both linear (LUAS-CMV) and circular (CUAS-CMV) forms of the UAS-CMV enhancer were successfully recruited to target genes by the dCas9-VP64-fused GAL4 protein.When co-transfected with a reporter gene in 293T cells, both LUAS-CMV and CUAS-CMV significantly activated gene expression on opposite of control conditions.

(Xu X, Gao J, Dai W, Wang D, Wu J, Wang J. Gene activation by a CRISPR-assisted trans enhancer. Elife. 2019 Apr 11;8:e45973. doi: 10.7554/eLife.45973. PMID: 30973327; PMCID: PMC6478495.)

This figure show Flow cytometry analysis of ZsGreen expression shows very high gene expression with (dCas9-VP64-GAL4/sgRNA-LUAS-CMV) and (dCas9-VP64-GAL4/sgRNA- CUAS-CMV) .

This figure shows graphic illustration of flow cytometry analysis of 293 T cells which shows high gene expression with both LUAS-CMV and CUAS-CMV. These findings highlight the potential of the GAL4-UAS system for efficient CRISPR-mediated trans-enhancer activation .

Also these vectors ( dCas9-VP64-GAL4 with linear (LUAS-CMV) and circular (CUAS-CMV) CMV enhancer) were constructed and co-transfected with a reporter gene(HNF4α gene) in HepG2 cells . then results were illustrated and compared with the results of the same construcrts in 293T cells.

(Xu X, Gao J, Dai W, Wang D, Wu J, Wang J. Gene activation by a CRISPR-assisted trans enhancer. Elife. 2019 Apr 11;8:e45973. doi: 10.7554/eLife.45973. PMID: 30973327; PMCID: PMC6478495.)

This figure shows graphic illustration of flow cytometry analysis of 293 T cells and HepG2 cells which shows high gene expression with (dCas9-VP64-GAL4/sgRNA-LUAS-CMV) and (dCas9-VP64-GAL4/sgRNA- CUAS-CMV). Gene expression was higher in HepG2 cells. These findings highlight the potential of the GAL4-UAS system for efficient CRISPR-mediated trans-enhancer activation .

Experimental Characterization

we digested dCas9(C)_NLS-Syn-VEGFR-1 grafetd with UAS Trans CMV enhancer and performed gel electrophoresis to detect the digested fragment.

This figure illustrates the digested dCas9(C)_NLS-Syn-VEGFR-1 prior to insert ligation .

Literature Characterization

Three AAV-2 vectors were created by inserting different promoters (CMV enhancer, PDGF, or a hybrid of the two) into a plasmid containing a firefly luciferase gene. These plasmids were then combined with two other plasmids (pAAV-RC and pHelper) and introduced into HEK293 cells which resulted in the production of three AAV-2 vectors, named AAV-CMV enhancer-luc, AAV-PDGF-luc, and AAV-CMV enhancer/PDGF-luc. The researchers tested how well the three AAV-2 vectors infected different types of cells, including primary rat brain cells (neurons and glial cells), and mouse and human cell lines (C17.2 and NT2-N) that had been grown to become neurons. They used a high dose (MOI of 1000) to ensure maximum infection.

AAV-2 vectors containing a hybrid CMV enhancer/PDGF promoter significantly increased luciferase gene expression in primary cortex neurons compared to vectors with the CMV enhancer/P promoter or the PDGF promoter alone respectively, at both 4 and 7 days post-transduction (Figures 1A and 1B).. This effect was seen in both mouse and human neuronal cells. However, in primary glial cells, the CMV enhancer/P promoter was more effective than the hybrid promoter(Figure 1C). These results suggest that the hybrid promoter retains its neuronal specificity.

ٌReference

Wang, C. Y., Guo, H. Y., Lim, T. M., Ng, Y. K., Neo, H. P., Hwang, P. Y. K., ... & Wang, S. (2005). Improved neuronal transgene expression from an AAV‐2 vector with a hybrid CMV enhancer/PDGF‐β promoter. The Journal of Gene Medicine: A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications, 7(7), 945-955.‏

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]