Difference between revisions of "Part:BBa K5036005"

Latest revision as of 13:08, 2 October 2024

VP64

Part Description

It is a transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16L) connected with glycine-serine (GS) linkers. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator

Usage

This part is fused with GAL4, dcas9(c), UAS Trans CMV enhancer. 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 VP64 which is linked to dCas9(c) in dCas9(C)-TF-synVEGFR1 receptor's first chain .

Characterization by Mathematical Modeling

The model provides the activation kinetics of VP64 transcription activator which occurs subsequent to 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 VP64 activation based on parametric values from literature .

Graph(1). Illustrates the relation between activation level of VP64 transcription activator (Yellow 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 .

we digested dCas9(C)_NLS-Syn-VEGFR-1 grafetd with VP64 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

vp64 were transfected into HEK293T reporter cells and treated with abscisic acid or DMSO for 48 h.

Statistical significance was calculated with one-way ANOVA, using Dunnett’s multiple testing correction which showed that GFP expression was higher with VP64 than with any other promoter.

Reference

Morita S, Horii T, Kimura M, Hatada I. Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform. Int J Mol Sci. 2020 Feb 25;21(5):1574. doi: 10.3390/ijms21051574. PMID: 32106616; PMCID: PMC7084704.

(Alerasool, N., Leng, H., Lin, Z. Y., Gingras, A. C., & Taipale, M. (2022). Identification and functional characterization of transcriptional activators in human cells. Molecular cell, 82(3), 677-695.)

Sequence and Features