TY - JOUR
T1 - Efficient and reproducible multigene expression after single-step transfection using improved bac transgenesis and engineering toolkit
AU - Zhao, Binhui
AU - Chaturvedi, Pankaj
AU - Zimmerman, David L.
AU - Belmont, Andrew S.
N1 - Funding Information:
This work was supported by the National Institute of General Medical Sciences (GM098319 and, in part, GM58460 to A. S. B.) and by the National Institutes of Health Common Fund 4D Nucleome Program (U54 DK107965 to A. S. B). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. We thank Edith Heard (Curie Institute) for providing DHFR BAC (clone 057L22 from CITB mouse library), Veena K. Parnaik (CSIR-CCMB, Hyderabad, India) for GFP-lamin B1 plasmid, Miroslav Dundr (Rosalind Franklin University of Medicine and Science) for GFP-fibrillarin plasmid, Huimin Zhao (University of Illinois Urbana–Champaign) for pQCXIN-TetR-mCherry plasmid, Peter Adams (Sanford Burnham Prebys Medical Discovery Institute) for BJ-hTERT cells, and K. V. Prasanth (University of Illinois Urbana–Champaign) for mRFP-magoh plasmid. The use of the BD FACS AriaII was assisted by the flow cytometry facility equipment at the Roy J. Carver Biotechnology Center, University of Illinois at Urbana–Champaign (UIUC).
Publisher Copyright:
© 2020 American Chemical Society.
PY - 2020/5/15
Y1 - 2020/5/15
N2 - Achieving stable expression of a single transgene in mammalian cells remains challenging; even more challenging is obtaining simultaneous stable expression of multiple transgenes at reproducible, relative expression levels. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC "scaffold" containing the mouse Msh3-Dhfr locus (DHFR BAC). Here, we extend this "BAC TG-EMBED" approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01- to 5-fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes. Second, we demonstrate little variation in expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making the choice of BAC scaffolds more flexible. Third, we present a novel BAC assembly scheme, "BAC-MAGIC", for inserting multiple transgenes into BAC scaffolds, which is much more time-efficient than traditional galK-based methods. As a proof-of-principle for our improved BAC TG-EMBED toolkit, we simultaneously fluorescently labeled three nuclear compartments at reproducible, relative intensity levels in 94% of stable clones after a single transfection using a DHFR BAC scaffold containing 4 transgenes assembled with BAC-MAGIC. Our extended BAC TG-EMBED toolkit and BAC-MAGIC method provide an efficient, versatile platform for stable simultaneous expression of multiple transgenes at reproducible, relative levels.
AB - Achieving stable expression of a single transgene in mammalian cells remains challenging; even more challenging is obtaining simultaneous stable expression of multiple transgenes at reproducible, relative expression levels. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC "scaffold" containing the mouse Msh3-Dhfr locus (DHFR BAC). Here, we extend this "BAC TG-EMBED" approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01- to 5-fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes. Second, we demonstrate little variation in expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making the choice of BAC scaffolds more flexible. Third, we present a novel BAC assembly scheme, "BAC-MAGIC", for inserting multiple transgenes into BAC scaffolds, which is much more time-efficient than traditional galK-based methods. As a proof-of-principle for our improved BAC TG-EMBED toolkit, we simultaneously fluorescently labeled three nuclear compartments at reproducible, relative intensity levels in 94% of stable clones after a single transfection using a DHFR BAC scaffold containing 4 transgenes assembled with BAC-MAGIC. Our extended BAC TG-EMBED toolkit and BAC-MAGIC method provide an efficient, versatile platform for stable simultaneous expression of multiple transgenes at reproducible, relative levels.
KW - Bac recombineering
KW - Copy-number-dependent expression
KW - Gene silencing
KW - Multireporter
KW - Promoter toolkit
KW - Transgene expression
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U2 - 10.1021/acssynbio.9b00457
DO - 10.1021/acssynbio.9b00457
M3 - Article
C2 - 32216371
AN - SCOPUS:85085264620
SN - 2161-5063
VL - 9
SP - 1100
EP - 1116
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 5
ER -