Belmont Lab
 Unversity of Illinois at Urbana-Champaign
Alumni Labs
 
Improving transgene expression in mammalian cells

Expression of transgenes in mammalian cells is an enormously important technology in a wide range of research fields from biotechnology to medical and basic cell biological research. Two major problems are gene delivery and gene expression. In general transgene technology still struggles with obtaining reproducible, stable expression of transgenes at levels comparable to endogenous genes. Chromosome "position effects" typically restrict transgene expression to much lower levels than endogenous gene counterparts. In addition expression is often lost with repeated cell passaging or with differentiation to another cell type.

In our studies of large-scale chromatin structure using multi-copy insertions of bacterial artificial chromosomes (BACs) carrying large mouse or human genomic DNA inserts, we noticed the ability of certain BAC constructs to form chromosome regions with "open" large-scale chromatin structures, independent of their chromosomal insertion sites. We hypothesized that this open large-scale chromatin conformation might create a permissive environment for reproducible transgene expression.

This hypothesis was tested by inserting reporter mini-genes expressing fluorescent proteins driven by common, viral promoters. We inserted fluorescent GFP or mRFP reporter mini-genes driven by viral promoters at different locations within a BAC containing a 170 kb region containing the mouse DHFR locus. This BAC was then transfected into mouse 3T3 cells, and stable colonies isolated containing the BAC transgenes inserted at many different chromosomal positions. Using flow cytometery we demonstrated a remarkably linear, copy number dependent, position independent expression for the reporter genes. We also demonstrated the capability of this approach to produce reproducible, copy number dependent, position independent expression of multiple reporter genes.

Results using this "BAC embedding" method for transgene expression were superior to a now standard method of flanking transgenes with the chicken core HS4 insulator sequences. Current research is continuing in order to further improve this technology and apply it to the general problem of multi-gene transgenesis.

Publication:

Bian Q, Belmont AS. BAC TG-EMBED: one-step method for high-level, copy-number dependent, position-independent transgene expression. Nucleic Acids Res. 2010 Jun 1; 38 (11) :e127. PubMed PMID:20385594; PubMed Central PMCID: PMC2887973.

 

 

 

 

 

 

 

 

 

 

       
 
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