A Nanobody-Based System Using Fluorescent Proteins as Scaffolds for Cell-Specific Gene Manipulation

Abstract

Fluorescent proteins are commonly used to label cells across organisms, but the unmodified forms cannot control biological activities. Using GFP-binding proteins derived from Camelid antibodies, we co-opted GFP as a scaffold for inducing formation of biologically active complexes, developing a library of hybrid transcription factors that control gene expression only in the presence of GFP or its derivatives. The modular design allows for variation in key properties such as DNA specificity, transcriptional potency, and drug dependency. Production of GFP controlled cell-specific gene expression and facilitated functional perturbations in the mouse retina and brain. Further, retrofitting existing transgenic GFP mouse and zebrafish lines for GFP-dependent transcription enabled applications such as optogenetic probing of neural circuits. This work establishes GFP as a multifunctional scaffold and opens the door to selective manipulation of diverse GFP-labeled cells across transgenic lines. This approach may also be extended to exploit other intracellular products as cell-specific scaffolds in multicellular organisms.

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

“…Finally, associating efficient localised electroporation with the rapidly evolving fluorescent protein-based tools for optogenetics (Nowotschin and Hadjantonakis, 2009;Tang et al, 2013;Yin and Wu, 2013) will surely provide new powerful approaches for the investigation of the cellular bases of morphogenesis.…”

Vectors and Gene Delivery to the Retina

“…For example, coinjection of AAVs transporting an inverted optogene sequence flanked by two loxP sites in a mouse line expressing cre in a subset of retinal neurons would allow specific delivery of the optogene into the retinal circuit. Furthermore, transgenic GFP reporter lines can be used for functional studies because of a method that transforms GFP into a molecule that enables genetic manipulation (Tang et al 2013(Tang et al , 2015. In this system, a cre recombinase dependent on GFP (cre-DOG) is used to directly induce cre/loxP recombination in GFP-expressing cells.…”

“…Both GFP (Tang et al 2013) and cre (Wang 2009) can be used in combination with viral vectors to confine expression of optogenetic actuators or sensors in the retina in a circuit-specific manner. A recombination event between two DNA recognition sites called loxP sites is catalyzed by crerecombinase.…”

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

“…As a result, the GFP expression in GFP-expressing cells could specifically lead to the formation of a nanoscaled active transcription factor. 54 This approach could be utilized to conveniently induce cellspecific transgene expression or gene knockdown by RNAi in a vast collection of transgenic GFP cell lines. 2 …”

“…2 Recently, some other elegant works also demonstrated a nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation. 54 Two different nanobodies that could bind different regions of GFP were fused to a transcriptional activation domain or a DNA-binding domain, respectively. As a result, the GFP expression in GFP-expressing cells could specifically lead to the formation of a nanoscaled active transcription factor.…”