InXy and SeXy, compact heterologous reporter proteins for mammalian cells

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.     

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform

Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.     

Orthogonal control of endogenous gene expression in mammalian cells using synthetic ligands

Gene switches have wide utility in synthetic biology, gene therapy, and developmental biology, and multiple orthogonal gene switches are needed to construct advanced circuitry or to control complex phenotypes. Endogenous vascular endothelial growth factor (VEGF‐A) is crucial to angiogenesis, and it has been shown that multiple alternately spliced VEGF‐A isoforms are necessary for proper blood vessel formation. Such a necessity limits the utility of direct transgene delivery, which can provide only one splice variant. To overcome this limitation, we constructed a gene switch that can regulate the (VEGF‐A) locus in mammalian cells by combining an engineered estrogen receptor (ER) ligand‐binding domain (LBD), a p65 activation domain, and an artificial zinc‐finger DNA binding domain (DBD). Our gene switch is specifically and reversibly controlled by 4,4′‐dyhydroxybenzil (DHB), a small molecule, non‐steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, an endogenous VEGF‐A induction ratio of >100‐fold can be achieved in HEK293 cells at 1 µM DHB, which is the highest endogenous induction reported to date. In addition, induction has been shown to be reversible, repeatable, and sustainable. Another advantage is that the ligand response is tunable by varying the clonal composition of a stably integrated cell line. The integration of our findings with the technology to change ligand specificity and DNA binding specificity will provide the framework for generating a wide array of orthogonal gene switches that can control multiple genes with multiple orthogonal ligands. Biotechnol. Bioeng. 2013; 110: 1419–1429. © 2012 Wiley Periodicals, Inc.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

Overview of vector design for mammalian gene expression

The expression of cloned genes in mammalian cells is a basic tool for understanding gene expression, protein structure, and function, and biological regulatory mechanisms. The level of protein expression from heterologous genes introduced into mammlaian cells depends upon multiple factors including DNA copy number, efficiency of transportation, mRNA processing, mRNA transport, mRNA stability, and translational efficiency, and protein processing, transport, and stability. Different genes exhibit different rate limiting steps for efficient expression. Multiple strategies are available to obtain high level expression in mammalian cells. This article reviews vector design for expression of foreign genes in mammalian cells.     

Construction of adenovirus for high level expression of small RNAs in mammalian cells

A series of plasmid vectors have been generated to allow the rapid construction of adenoviral vectors designed to express small RNA sequences. A truncated human U6 gene containing convenient restriction sites has been shown to be expressed at high levels following electroporation into a series of human cell lines. This gene was ligated into a promoterless adenoviral plasmid, and we have generated high titer virus by homologous recombination with adenoviral Addl327 DNA in 293 cells. Recombinant adenovirus containing a hammerhead ribozyme sequence targeted toward the Bcl-2 mRNA has been used to transduce a panel of human tumor cell lines. We have demonstrated high level expression of the recombinant U6 gene containing the ribozyme and reduction of Bcl-2 protein in transduced cells. These plasmids are suitable for the development of adenoviral vectors designed to express both ribozymes and antisense RNA in human cells.     

Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering

The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering.