Novel Technologies and Tools developed in the Blau Lab
Bioengineeering of stem cell nichesTo enhance our understanding of stem cell biology, our laboratory is engineering adult stem cell niches using a versatile platform technology to form synthetic biomolecular hydrogels with well-defined biochemical and structural properties. This promising family of cellular scaffolds was first developed by Dr. Matthias Lutolf and coworkers in the world renowned laboratory of Prof. Jeffrey Hubbell at the ETH Zurich and EPF Lausanne, Switzerland. These scaffolds consist of two major components that are mixed to form a three-dimensional hybrid network under physiologic conditions: i) hydrophilic multifunctional poly(ethylene glycol) macromers (PEG, a non-toxic polymer that can be attached to proteins without impairing their bioactivity) and ii) bioactive oligopeptide or protein components containing reduced thiols from cysteine residues. By employing heterofunctional PEG linkers bearing an active ester, virtually any biomolecule can be tethered to the matrix via its amine groups. Using this chemical scheme we can generate artificial signaling microenvironments with tailor-made compositions (and concentrations) of potential stem cell regulatory proteins. Our goal is to deconstruct and reconstruct the complexity of natural stem cell niches in vitro in order to elucidate the control mechanisms governing self-renewal and differentiation.
Quantitative Detection of Inducible Protein Translocation Using Proximity-Based Enzyme Complementatio(Wehrman et al, Nature Methods (2005). Patents: Stanford docket S03-048, Stanford docket S96-125/US 6,342,345, Stanford docket S96-125/US Appl US-2002-0048778-A1, Stanford Docket S02-123/US Appl US-2003-0175836-A1)
Fundamental to eukaryotic cell signaling is the regulation of protein translocation which has been largely qualitative, due to the limitations of existing technologies. We originally used a β-galactosidase complementation technology to assay cell fusion. More recently, our laboratory generated novel complementing mutant peptides which complement weakly to assess protein translocation based on proximity. The Complementation Assay for Protein Translocation (CAPT) is derived from β-galactosidase and is comprised of one enzyme fragment (ω) that is localized to a particular subcellular region, and a small complementing mutant peptide (α) that is fused to the protein of interest. The concentration of α in the immediate vicinity of ω correlates with the amount of enzyme activity obtained in a dose and time dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane can now be quantitatively monitored in multi-well format and in live mammalian cells by flow cytometry. Examples include the glucocorticoid receptor, the C1A domain of PKC gamma, and the PH domain of AKT. This system allows resolution of signaling pathways and validation of screens for molecules that promote or disrupt protein translocation.
Luminescent imaging of β-galactosidase activity in living subjects using sequential reporter enzyme luminescence (SRL) (Wehrman et al, Nature Methods (2006). Patent: Stanford docket S03-048/US Appl. 11/132,764)
We generated a Sequential Reporter enzyme Luminescence (SRL) technology for the in vivo detection of β-galactosidase activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by β-galactosidase before it can be catalyzed by firefly luciferase to generate light. As a result, luminescence is dependent on β-galactosidase activity. Constitutive β-galactosidase activity in engineered cells and inducible tissue specific β-galactosidase expression in transgenic mice can now be visualized non-invasively over time. Another substantial advantage of using β-galactosidase as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike firefly luciferase which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant β-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, using SRL, the luminescent properties of firefly luciferase can be coupled to the advantages of other enzymes enabling bioluminescent imaging applications that were previously not possible.
Detection of Protein-Protein Interactions and Families of Membrane Receptors by beta-galactosidase complementation (patent and two publications pending)
ErbB2 Family of Receptors: Herceptin (Trastuzumab), an antibody to ErbB2 (HER2/Neu), reduces recurrence and improves survival in a subset of ErbB2 positive breast cancer patients, yet its effects on EGF receptor dimerization remain poorly understood. Using a new method for monitoring the dynamic interactions of membrane receptors in a quantitative manner, we found that Herceptin specifically reduces heterodimer levels. These results suggest that novel pharmacological agents for treatment of ErbB2-positive carcinomas should be screened for their effects on ErbB family dimerization patterns.
TrkA and p75 Receptors: Nerve growth factor binds and activates two structurally distinct transmembrane receptors, TrkA and p75. These receptors have been proposed to cooperate to form the classical “high affinity” NGF binding site through the formation of a heterotrimeric TrkA/NGF/p75 complex. In order to define a structural basis for the high affinity NGF binding site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. We also investigated the hetero-dimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a novel protein-protein interaction system. The results suggest that these receptors do not cooperate to form a high affinity NGF binding site. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and shared adaptor molecules, and not through direct protein interactions.
Restriction Enzyme Generated siRNA (REGS) Vectors and Libraries (Sen, G, et al, Nature Genetics (2004). Patent: Stanford docket S03-243.)
This methodology is the first to allow genome wide screens of unbiased siRNAs to all genes and untranslated regions. This novel method was designed for generating numerous siRNA constructs from any gene of interest or pool of genes using a combination of restriction enzyme digests and hairpin loop ligations. Small interfering RNA (siRNA) technology greatly facilitates loss of gene function studies in mammalian cells. However, the generation of multiple unique siRNA expression vectors is slow, inefficient and costly. To test if the Restriction Enzyme Generated siRNAs (REGS) generated were functional, a transgene and two endogenous genes were silenced, resulting in the predicted phenotypes. REGS generates approximately 34 unique siRNAs per kilobase of sequence with greater than 96% of the cloned inserts containing siRNAs with the appropriate structure. The efficiency of REGS enabled the creation of siRNA libraries from double stranded cDNA containing 105–106 independent clones. The high yield of siRNAs per gene and the creation of a highly complex siRNA library facilitated by REGS is unprecedented. This loss of function technology will be invaluable in determining prerequisites for nuclear reprogramming and components of key signaling pathways. Dr. Blau has disseminated the technology developed in her laboratory licensing it to a Biotechnology company, which sells it as a kit, facilitating its use worldwide.
Regulatable Gene Expression Vectors: (Rossi, F.M.V., et al,Nature Genetics (1998) Kringstein, A.M, et al,PNAS (1998); Rossi, F.M.V., et al, Molecular Cell (2000). Patent: Stanford docket ???)
The tetracycline inducible retroviral vectors developed in our laboratory allow rapid delivery and fine control of gene expression in mammalian cells in tissue culture. By engineering different dimerization domains into transcriptional activators and repressors, expression of both types of molecules in the same cell is not possible, a system designated RetroTet-ART (Retroviral Tetracycline-inducible Activators and Repressors Together). As a result, gene expression can be completely repressed and then induced up to six orders of magnitude. Using this system, we showed that highly conserved 3’untranslated sequences are key post-translational regulatory elements that control protein levels by altering stability. We also demonstrated that increasing concentrations of extracellular signals leads to graded rheostat-like transcriptional responses when either activators or repressors are expressed alone. However, the expression of both activators and repressors together leads to an all-or-none switch-like response. These findings explain why in nature, stripes with sharp borders are found during Drosophila development and why cells generally avoid neoplasia by shifting completely from quiescence to growth.
Muscle mediated gene delivery (Dhawan, J, et al,Science (1991); Gussoni, E, et al,Nature (1992); Kang, S-M., et al,Science (1997); Gussoni, E, et al,Nature Medicine (1997); Springer, M.L, et al, Molecular Cell (1998); Ozawa, C.R., et al,JCI (2004). Patents: Stanford docket S89-054/US 5,538,722 and VIP patent disclosure.)
Our laboratory first showed that muscle is an ideal target for delivery of genes encoding products secreted locally in muscle tissues and into the circulation. Muscle is now used as the tissue of choice for a range of gene therapy vectors encoding secreted proteins. We found that human growth hormone could be secreted at physiological levels by genetically engineered myoblasts implanted into the muscles of mice. Subsequent studies showed that Fas ligand secreted by genetically engineered myoblasts induced an immune response and inhibition of solid tumor growth. In clinical trials of Duchenne muscular dystrophy patients, we demonstrated that human myoblast mediated cell therapy was safe and led to the localized expression of the missing dystrophin transcripts and proteins in patients. Currently, this technology is being used in studies of principles of angiogenesis with a view toward clinical applications for therapeutic blood vessel development in ischemic cardiac and skeletal muscle.
Our laboratory has provided these technologies and vectors to hundreds of laboratories worldwide.
Mutation of the β-galactosidase α peptide to generate weakly complementing mutants.
Using the wild-type high-affinity α-peptide, we have created a system that is ideally suited to monitoring translocation events that occur between subcellular compartments such as the cytosol and nucleus. However, application of this system to protein movement within the same subcellular compartment failed due to the high level of spontaneous complementation that occurs when both enzyme portions are expressed in the same physical space. To generate a system more suitable for translocation within the same subcellular compartment, we reasoned that mutants with higher dissociation rates and thus weaker complementation abilities were required. Several α point mutants were generated based on the interaction domains of α and ω. These peptides complemented ω to different degrees resulting in enzyme activities that varied over a 30-fold range. Picture represent the Crystal structure of β-galactosidase with the α portion pictured in yellow and arrows indicate point mutations. Weakly complementing α peptides force enzyme complementation through increases in local concentration.
Enzyme complementation is defined as the ability of two inactive enzyme fragments to come together and restore enzyme activity. We have adapted intracistronic complementation of the Escherichia coli lacZ gene for use in mammalian cells. β-galactosidase activity detectable by quantitative biochemical assay, flow cytometry, or microscopy is produced from the forced interaction of nonfunctional weakly complementing β-galactosidase peptides and serves as a measure of the extent of interaction of the non-β-galactosidase portions of the chimeras. This approach allows a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.
Nuclear translocation measured using high affinity β-galactosidase complementation
Gain of signal nuclear translocation assay. The ω fragment (blue) is localized to the nucleus with a nuclear targeting signal (red) and the cytosolic protein of interest (yellow) is fused to the minimal α peptide (black). Upon stimulation, the α fusion moves to the nucleus and complements ω, increasing β-galactosidase activity. The bottom-right panel shows a time course of Dex induced nuclear translocation of the GR assayed by increases in β-galactosidase activity. Loss of signal assay. The ω fragment (blue) is tethered to the plasma membrane using the extracellular and transmembrane regions of the EGFR. In this system the cytosolic α-fusion complements spontaneously to high levels. Nuclear translocation of the α fusion results in a loss of enzyme activity due to limited concentrations of the two fragments. The bottom-left panel depicts the loss of β-galactosidase activity associated with Dex induced translocation of the GR from the cytoplasm to the nucleus.
In vivo Sequential Reporter enzyme Luminescence Imaging (SRL)
We have developed a technology to image Beta-galactosidase activity using luminescence in living mice. Mouse implanted with cell expressing luciferase only (left leg) or luciferase+Beta-gal (right Leg). Left panel: Injection of the substrate Lugal reveals Beta-gal activity in the right leg but only minimal luminescence over the left leg. Right panel: Injection of luciferin does not differentiate between cells that express Beta-gal and controls, showing luminescence over both legs.