A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

Abstract

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.

Antibody expression stability in CHO clonally derived cell lines and their subclones: Role of methylation in phenotypic and epigenetic heterogeneity

“…The EF‐1α promoters have primarily been studied for transgene expression in gene therapy vectors, showing stable expression in proliferating cultures compared to CMV promoters . In CHO cells, Spencer et al introduced single‐copy GFP transgenes driven by minimal CMV and EF‐1α promoters and found that EF‐1α clones were less stable than the CMV clones.…”

Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

“…AAV-based gene targeting assays (an assay based on the Hyg R –5′ EGFP reporter system as well as assays targeting PIGA exon 6 and PIGA intron 5, respectively) were performed as previously described with some modifications ( 10 , 11 , 38 ). For all AAV-based gene targeting assays, AAV particles (serotype 2) were produced by co-transfection of HEK293T cells with AAV-based targeting plasmids along with the plasmids pAAV-RC and pHelper included in the AAV Helper-Free System (Agilent Technologies).…”

Gene Delivery and Expression Systems in Induced Pluripotent Stem Cells

“…This unique property permits gene editing, and effi cient gene targeting by AAV vectors has been achieved in human ES cells and iPS cells [ 34 -36 ]. Damdindorj et al [ 37 ] reported that when used in an AAV vector, the CMV promoter provided stable and robust gene expression in cancer cell lines; however, this promoter does not seem to be preferable for noncancerous cell lines and for the purpose of AAV-based gene targeting. Further studies are needed to identify the most suitable constitutive promoter for the application of AAV vectors to precise genetic manipulation of iPS cells, which could have great scientifi c and therapeutic potential.…”