Abstract
Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a β-catenin-ERα fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-β-catenin-ERα vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.
Highlights
The rapid development of genomic functional research requires stringently controlled expression systems
As a genomic DNA fragment with effective insulator characteristics, bacterial artificial chromosome (BAC) vectors enable the generation of transgenic mammalian cells with predictable expression characteristics independent of the integration site, so they are currently often used in genetic research, such as library construction, transgenic mice production, and gene targeting constructs [20,21]
Contruction of pIGR-reading frame C (RFC) RFC (Invitrogen, Calsbad, CA, USA), enhanced green fluorescent protein (EGFP), internal ribosome entry site (IRES) and all the elements of Tet-inducible expression system were cloned into pE11.F3.M.F
Summary
The rapid development of genomic functional research requires stringently controlled expression systems. As a genomic DNA fragment with effective insulator characteristics, BAC vectors enable the generation of transgenic mammalian cells with predictable expression characteristics independent of the integration site, so they are currently often used in genetic research, such as library construction, transgenic mice production, and gene targeting constructs [20,21]. The gene of interest is cloned into an entry vector (pENTR), which is mixed in vitro with pIGR-RFC and the Gateway Clonase enzyme mixtures.
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