A System for Creating Stable Cell Lines that Express a Gene of Interest from a Bidirectional and Regulatable Herpes Simplex Virus Type 1 Promoter

<div><p>Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the <i>Sleeping Beauty</i> transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models.</p></div

Gene expression levels achieved using the inducible, HSV IE bidirectional promoter.

<p>MFI, mean fluorescence intensity. Values are mean ± s.e.m., n = 3 experiments for two independent cell lines.</p><p>Gene expression levels achieved using the inducible, HSV IE bidirectional promoter.</p

Phenotypes of cell lines generated using a commercially available Tet-On vector system and <i>Sleeping Beauty</i> transposon-mediated integration.

<p>^apercent of doxycycline induced cells per clone.</p><p>MFI, mean fluorescence intensity. Values for No Dox, Plus Dox, and Fold Induction are mean ± s.e.m.</p><p>Phenotypes of cell lines generated using a commercially available Tet-On vector system and <i>Sleeping Beauty</i> transposon-mediated integration.</p

Phenotypes of cell lines generated using the inducible HSV1-IE bidirectional promoter and <i>Sleeping Beauty</i> transposon-mediated integration.

<p>^apercent of doxycycline induced cells per clone.</p><p>MFI, mean fluorescence intensity. Values for No Dox, Plus Dox, and Fold Induction are mean ± s.e.m.</p><p>Phenotypes of cell lines generated using the inducible HSV1-IE bidirectional promoter and <i>Sleeping Beauty</i> transposon-mediated integration.</p

The Tet-responsive HSV-IE promoter is tightly controlled and provides a broad range of gene expression.

<p>(A) Diagram of controlled and dynamic changes in gene expression levels achieved using the modified HSV IE bidirectional promoter. Transposon gene transfer is used to simultaneously create a cell line with: i) stable expression of the tetracycline repressor protein (TetR; filled circle) and ii) constitutive expression of NGFR coupled with inducible expression of the gene of interest (GOI) controlled by the inducible IE bidirectional promoter. In the repressed state, TetR proteins bind to the target sequences (2xOp) and inhibit transcription of the GOI (OFF). TetR is inhibited upon addition of doxycycline (Dox) and transcription activated (De-Repressed, ON). Transcriptional activity of the IE promoter and expression of the GOI can be further enhanced, only for de-repressed cells, upon expression of VP16 transactivator (Induced). (B) Schematic diagram of SB transposon vectors encoding for the HSV-IE promoter with tandem copies of tetracycline-repressor target sequences (2xOp) introduced near the TATA site (G-IE-N (TR^TATA). (C) Overlay of flow cytometry histograms demonstrating GFP expression for control cells (red), or a representative cell line cultured in the absence of doxycycline (blue, repressed), presence of doxycycline (green, de-repressed), or when doxycycline treated cells were transduced with adenovirus vector particles (m.o.i. = 3) encoding for expression of VP16 (pink, induced). (D) Fluorescent microscopy images of the same cell lines demonstrating GFP expression in the indicated states. (E) Dot plots for the same clone demonstrating coexpression of GFP and NGFR by flow cytometry in the repressed, de-repressed and induced states. VP16, trans-activator of HSV immediate early gene expression; RE, responsive element; NGFR, nerve growth factor receptor; GFP, green fluorescent protein; pA, BGH polyadenylation signal; A; SV40 polyadenylation signal.</p

The CMV IE promoter demonstrates only unidirectional activity.

<p>(A) Schematic diagram of the inducible 728-bp CMV promoter (CMV-TRE, T-REx, Life Technologies) relative to a 2081-bp version of the CMV immediate early (IE) promoter. Closed arrow heads indicate direction of transcription, blue shading identifies consensus nucleotide sequences between the two elements where orange and green colored bars highlight canonical and non-canonical TATA boxes, respectively. The purple box for CMV-TRE identifies a 40-bp sequence encoding for two copies of a Tet-responsive element (TRE) that serve as binding sites for the Tet-repressor protein. The CMV IE sequence was inserted into a <i>Sleeping Beauty</i> transposon plasmid in between coding sequences for GFP and truncated nerve growth factor receptor (NGFR) by blunt cloning to create G-C-N. A scale bar is shown to estimate relative size of each promoter where (-) and (+) signs correspond to the reverse and forward directions. (B) Versions of G-C-N with the CMV promoter in both orientations are shown. Each version was cotransfected into human embryonic kidney (HEK-293T) cells to create puromycin-resistant cell clones (n = 5 per orientation). Representative dots plots demonstrating expression of NGFR and GFP by flow cytometry for the CMV IE promoter in forward and reverse orientations. CMV, cytomegalovirus; A, SV40 polyadenylation signal; pA, bovine growth hormone (BGH) polyadenylation signal.</p

Failure to consistently generate clonal cell populations with an “off/on” phenotype using a commercially available tetracycline inducible expression system.

<p>(A) Schematic diagram of the plasmids and methodology used to create blasticidin-resistant (Bsd^R) cell lines with stable expression of the tetracycline-repressor (Tet^R) protein. These cells were further modified for coexpression of a hygromycin-resistance gene (Hyg^R) and enhanced green fluorescent protein (GFP) that is repressed “off” but can become transcriptionally active “on” upon administration of doxycycline (Dox). (B) Flow cytometry histograms (left) and fluorescent microscopy images (right, original magnification 40x) demonstrating expression of GFP in cell lines cultured in the absence (purple, No Dox) and presence of 4 μM doxycycline (orange; Dox). Shown are representative examples of clones that were not inducible (Uninduced), were not adequately repressed (Leaky), were only partially induced (Heterogenous), or considered to be optimally repressed and induced (Optimal). The percentage of clones for each category are indicated (n = 21 cell lines). CMV, cytomegalovirus; Tet^R, tet-repressor protein; TRP, tet-responsive promoter; Bsd, blasticidin resistance gene; Hyg, hygromycin resistance gene; GFP, green fluorescent protein; pA, poly adenylation signal; A, poly adenylation signal.</p

Improved utility of the inducible, bidirectional system by transposon delivery of the tetracycline repressor protein.

<p>(A) <i>Sleeping Beauty</i> (SB) transposons encoding for inducible expression of influenza A virus hemagglutinin (HA) gene (HA-IE-N) or bicistronic expression of the tetracycline-repressor (TetR) and puromycin resistance gene (Puro) were cotransfected with SB transposase (PGK-SB11) into HeLa cells to create cell lines with regulated levels of HA. (B) Western blot of total cell lysates prepared from two cell lines cultured in the absence of doxycycline (repressed, R), presence of 4 μM doxycycline (de-repressed, DR), or when doxycycline treated cells were transduced with adenovirus vector particles (m.o.i. = 3) encoding for expression of VP16 (induced, IN). Membranes were reacted with antibodies to HA or GAPDH, which served as a loading control. Molecular weights (kDal) are indicated. PGK, human phosphoglycerate kinase promoter; Cags, chimeric CMV enhancer:chicken beta-actin promoter; ires, internal ribosome entry site; pA, BGH polyadenylation signal; A; SV40 polyadenylation signal.</p

The HSV-1 IE promoter provides coordinate and constitutive expression of two genes and can be induced by VP16.

<p>(A) Top, genetic organization of the HSV-1 genome demonstrating the long-repeated (R_L) and short-repeated (R_S) regions that regulate expression of the immediate-early (IE) genes, infected cell proteins 0 (ICP0) and 4 (ICP4). Also indicated are unique long (UL) and unique short (US) regions that encode for most of the early and late genes. Middle, enhanced view of the IE regulatory element located between the ICP0 and long-short spanning transcript (L/ST) genes and includes six VP16 responsive elements (VP16 REs 1–6) and two TATA boxes (black bars). Bottom, 1724-bp of sequences introduced into a <i>Sleeping Beauty</i> transposon between GFP and NGFR to create G-IE-N. (B) Photomicrographs showing immunofluorescence staining for NGFR (left panel, red), direct fluorescence for GFP (middle panel, green), and merged images showing coexpression (right panel). Original magnification 40x. (C) Top, flow cytometry histograms of the clone depicted above demonstrating GFP expression before (purple) and after induction with VP16 (orange) respective of naïve controls (grey); bottom, dot plots demonstrating coordinate expression of GFP and NGFR before (purple) and after induction with VP16 (orange). (D) Mean fluorescence intensity for GFP and NGFR before (No VP16) and after (VP16) induction. *<i>P</i><0.001 using Student’s <i>t</i>-test when compared to No VP16. (E) Fold increase in fluorescence intensity for GFP and NGFR after VP16 induction. Graphical representations of GFP fluorescence and fold increase were calculated from three cell lines and reported as mean <u>+</u> sem. VP16, transactivator of HSV immediate early gene expression; RE, responsive element; NGFR, nerve growth factor receptor; GFP, green fluorescent protein; pA, BGH polyadenylation signal; A; SV40 polyadenylation signal.</p

Transposon-mediated delivery of the tetracycline inducible cassette does not improve the ability to generate clonal cell populations with an “off/on” phenotype.

<p>(A) <i>Sleeping Beauty</i> (SB) transposons encoding for inducible expression of GFP (TRP-GFP) or resistance to puromycin (PGK-Puro) were cotransfected with SB transposase (PGK-SB11) into HEK-293T expressing the tetracycline-repressor protein (Tet^R) to create cell lines that express GFP upon administration of doxycycline (Dox). (B) Flow cytometry histograms demonstrating expression of GFP in cell lines cultured in the absence (purple, No Dox) or presence (orange, Dox) of 4 μM doxycycline. Shown are representative examples of clones that were Uninduced, Leaky, Heterogenous, or Optimal with the percentage of clones for each group indicated. (C) GFP fluorescence intensity in the repressed (No Dox) and de-repressed (Plus Dox) states. *<i>P</i> = 0.0074 using Student’s <i>t</i>-test when compared to repressed (No Dox). (D) Fold increase in GFP fluorescence intensity when de-repressed (Plus Dox). Graphical representations of GFP fluorescence and fold increase were calculated from 19 clonal lines and reported as mean <u>+</u> s.e.m. TRP, Tet-responsive promoter; PGK, human phosphoglycerate kinase promoter; GFP, green fluorescent protein; SB11, <i>Sleeping Beauty</i> transposase; Puro, puromycin resistance gene; Tet^R, Tet-repressor protein; pA, poly adenylation signal.</p