1001. Metabolic Biotinylation of Cell Surface Receptors for In Vivo Imaging
ABSTRACT We have developed a versatile, potent technique for imaging cells in culture and in vivo by expressing a metabolically biotinylated cell-surface receptor and visualizing it with labeled streptavidin moieties. The recombinant reporter protein, which incorporates a biotin acceptor peptide (BAP) between an N-terminal signal sequence and a transmembrane domain, (BAP-TM) was efficiently biotinylated by endogenous biotin ligase in mammalian cells with the biotin displayed on the cell surface. Tumors expressing the BAP-TM have high sensitivity for magnetic resonance and fluorescence tomographic imaging in vivo after intravascular injection of streptavidin conjugated to magnetic nanoparticles or fluorochromes, respectively. Moreover, streptavidin-horseradish peroxidase conjugates in conjunction with a peroxidase-sensitive gadolinium agent further increased and prolonged the magnetic resonance signal. This BAP-TM allows noninvasive real-time imaging of any cell type transduced to express this reporter protein in culture or in vivo.
Full-textDOI: · Available from: Jan Grimm, Sep 26, 2014
- SourceAvailable from: Zhiyi Liu
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- "Indirect MRI labeling involves genetic manipulations and enables the cell to use either endogenous or exogenous substrates. Methods of indirect MRI labeling include: (1) genes encoding cell surface receptors/ligands are expressed in the cell to bind MRI probes that have been functionalized with streptavidin or transferrin 62, 63; (2) an HSV thymidine kinase is introduced in the cell to trap thymidine analogues that are detectable by CEST MRI 64; (3) iron-binding proteins (e.g., ferritin) are expressed in the cell to capture endogenous irons, which can produce paramagnetic contrast signals 65, 66; and (4) proteins rich in amide protons (e.g., lysine-rich protein, LRP) are produced in the cell and can be readily detected by CEST MRI 67. To address the concern of intracellular stability of the LRP gene, a newer MRI reporter gene based on human protamine‑1 (hPRM1) has been developed 68. "
ABSTRACT: Despite the remarkable progress of adoptive T cell therapy in cancer treatment, there remains an urgent need for the noninvasive tracking of the transfused T cells in patients to determine their biodistribution, viability, and functionality. With emerging molecular imaging technologies and cell-labeling methods, noninvasive in vivo cell tracking is experiencing impressive progress toward revealing the mechanisms and functions of these cells in real time in preclinical and clinical studies. Such cell tracking methods have an important role in developing effective T cell therapeutic strategies and steering decision-making process in clinical trials. On the other hand, they could provide crucial information to accelerate the regulatory approval process on the T cell therapy. In this review, we revisit the advances in tracking the tumor-specific CTLs, highlighting the latest development in human studies and the key challenges.Theranostics 07/2014; 4(10):990-1001. DOI:10.7150/thno.9268 · 8.02 Impact Factor
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- "The AP tag may also be replaced with the biotin acceptor domain from Propionibacterium shermanii 1.3S- transcarboxylase (PSTCD), which is recognized and biotinylated by an endogenous biotin ligase in mammalian cells. A displayed protein containing PSTCD may be directly labeled with streptavidin conjugated to magnetic nanoparticles or fluorophores (Tannous et al. 2006). "
ABSTRACT: Streptavidin and its homologs (together referred to as streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the streptavidin-biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in streptavidin engineering and new applications based on the streptavidin-biotin interaction.Applied Microbiology and Biotechnology 09/2013; 97(21). DOI:10.1007/s00253-013-5232-z · 3.34 Impact Factor
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- "Since then, four main categories of MRI reporter genes have emerged. They involve increasing endogenous accumulation of iron 68-70, exploiting cell surface interactions 71, 72, harnessing enzymatic reactions 67, 73, 74, or employing chemical exchange saturation transfer (CEST) 75, 76. "
ABSTRACT: Being able to self-renew and differentiate into virtually all cell types, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have exciting therapeutic implications for myocardial infarction, neurodegenerative disease, diabetes, and other disorders involving irreversible cell loss. However, stem cell biology remains incompletely understood despite significant advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment all hamper the transition from laboratory animal studies to human clinical trials. Although traditional histopathological techniques have been the primary approach for ex vivo analysis of stem cell behavior, these postmortem examinations are unable to further elucidate the underlying mechanisms in real time and in vivo. Fortunately, the advent of molecular imaging has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest. This review summarizes various molecular imaging technologies and how they have advanced the current understanding of stem cell survival, biodistribution, immunogenicity, and tumorigenicity.Theranostics 04/2012; 2(4):335-45. DOI:10.7150/thno.3666 · 8.02 Impact Factor