Dataset Information
New function of a well-known promoter: Enhancer activity of minimal CMV promoter enables efficient dual-cassette transgene expression.
Ontology highlight
ABSTRACT: Background
Co-expression of multiple genes in single vectors has achieved varying degrees of success by employing two promoters and/or application of viral 2A-peptide or the internal ribosome entry-site (IRES). However, promoter interference, potential functional-interruption of expressed-proteins by 2A-generated residual peptides or weaker translation of IRES-mediated downstream genes has curtailed their utilization. Thus, there is the need for single vectors that robustly express multiple proteins for enhanced gene therapy applications.Methods
We engineered lentiviral-vectors for dual-cassette expression of green fluorescent protein and mCherry in uni- or bidirectional architectures using the short-version (Es) of elongation factor 1α (EF) promoter and simian virus 40 promoter (Sv). The regulatory function of a core fragment (cC) from human cytomegalovirus promoter was investigated with cell-lineage specificity in NIH3T3 (fibroblast) and hematopoietic cell lines U937 (monocyte/macrophage), LCL (lymphoid), DAMI (megakaryocyte) and MEL (erythroid).Results
The cC element in reverse-orientation not only boosted upstream Es promoter to levels comparable to full-length EF in DAMI, U937 and 3T3 cells, but also blocked the suppression of downstream Sv promoter by Es in U937 and 3T3 cells with further improved Sv activity in DAMI cells. Such lineage-restricted up-regulation is likely attributed to two protein-binding domains of cC and diverse expression of related factors in different cell types for enhancer and terminator activities, but not spacing function.Conclusions
Such a newly developed dual-cassette vector could be advantageous, particularly in hematopoietic cell-mediated gene/cancer therapy, by allowing for independent and robust co-expression of therapeutic gene(s) and/or a selectable gene or imaging marker in the same cells.
SUBMITTER: Boateng-Antwi MKA
PROVIDER: S-EPMC9639367 | biostudies-literature | 2021 Nov
REPOSITORIES: biostudies-literature
Publications
New function of a well-known promoter: Enhancer activity of minimal CMV promoter enables efficient dual-cassette transgene expression.
The journal of gene medicine 20210818 11
<h4>Background</h4>Co-expression of multiple genes in single vectors has achieved varying degrees of success by employing two promoters and/or application of viral 2A-peptide or the internal ribosome entry-site (IRES). However, promoter interference, potential functional-interruption of expressed-proteins by 2A-generated residual peptides or weaker translation of IRES-mediated downstream genes has curtailed their utilization. Thus, there is the need for single vectors that robustly express multi ...[more]