Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

Abstract

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals

“…Consequently, its avoidance is a matter of ongoing research ( 61 , 62 ). Leaky expression is thought to arise due to positional effects after genomic integration, for instance because of close proximity to genomic enhancer elements ( 63 ). Our pre-miR switch design operates at the RNA level and is therefore unaffected by such interference.…”

De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium

“…This was accomplished using a recently published site-specific ZFNmediated knockin strategy ( Fig. 3c and Table S1) (70,71). GALNT6 re-introduction rescued the phenotype, and the ⌬T6ϩT6 cells formed disorganized clusters of cells with multiple small actin-lined lumens (Fig.…”

“…The human colon adenocarcinoma LS174T cell line was chosen as a model system based on its phenotypic characteristics and stable genome (70,81). Cells were grown in 50% DMEM 1965 and 50% Ham's F12 supplemented with 10% FBS and 1% L-glutamine.…”

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

“…The design was built on the PrIITE platform, where the Tet-On 3G (Tet-On) transactivator gene and inducible gene of interest (GOI) are introduced at separate loci (43). We predicted that it would be possible to stably incorporate both the Tet-On 3G gene and the inducible GOI at the same locus without loss of performance, and we used a tandem KI strategy where the first cassette provides a landing pad for the second cassette.…”

“…HEK cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. ObLiGaRe-mediated KI was performed as described previously (43). In brief, ϳ60% confluent HEK⌬T2 (2A6) and HEK⌬T11 (7E5) cells in 6-well plates were transfected with GFP/E2 Crimson-tagged AAVS1 ZFN plasmids and pAAVS1-EPB71-TETOn3G-ObLiGaRe donor construct in a ratio of 1:1:3 g, using Lipofectamine 3000 according to the manufacturer's instructions.…”