Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED

Abstract

Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the "BAC TG-EMBED" method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum-induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.

Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

“…Specifically, we measured the expression as a function of copy number of one specific reporter gene construct inserted into these BAC scaffolds. Previous applications of BAC TG-EMBED showed a linear relationship between copy number and expression level, largely independent of the chromosome integration site, demonstrating copy-number dependent, position independent transgene expression (51, 54). For active chromosomal regions, we chose the RP11-138I1 BAC containing the human ubiquitin B gene locus (UBB BAC), the RP23-401D9 BAC containing the “safe-haven” mouse Rosa26 genetrap locus (ROSA BAC) (91), and the CITB-057L22 BAC carrying the mouse Dhfr gene locus (DHFR BAC).…”

“…We previously demonstrated the feasibility of the BAC TG-EMBED approach using both the DHFR BAC (51) and a BAC containing the human GAPDH gene locus (GAPDH BAC) (54). We set out to extend this BAC TG-EMBED methodology in two new directions (Figure 1).…”

“…We had not previously observed such heterogeneous expression profile using our original DHFR BAC containing the CMV-driven mRFP alone or both the CMV-driven EGFP and CMV-driven mRFP reporter genes (51). However, we had observed ∼80% uniform clones for a GAPDH BAC scaffold with the UBC-GFP-ZeoR reporter gene inserted (54). The percentage of clones showing such heterogeneous expression varied from 58% to 83% for the 5 BAC scaffolds surveyed here (Table 1).…”

“…Thus, BACs with no transcription units would be more desirable. Another improvement over our previous BAC TG-EMBED system (51, 54) would be a toolkit of endogenous promoters capable of driving transgene expression over a wide range of defined, relative expression levels. Viral promoters, including the CMV promoter we used previously, are known to be prone to epigenetic silencing (55, 56), while most previously used endogenous and synthetic promoters were selected for their strength (53, 57–60).…”

Redesigned Reporter Gene for Improved Proton Exchange-based Molecular MRI Contrast

“…Stability and reproducibility are two essential factors required for the successful application of a reporter gene 35 . Furthermore, to allow the correct quantification of the biological/therapeutic process of interest (e.g., the extent of viral replication and spread during oncolytic virotherapy 5 ), it is essential to reliably know the exact composition of the contrast-generating substance in the reporter (e.g., the number of amide bonds of the backbone, as affected by the environment induced by the presence of lysine amino acids) and avoid contaminating effects, such as random protein truncation.…”