Cepko, Constance

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Cepko, Constance

cepko@genetics.med.harvard.edu

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Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies
(eLife Sciences Publications, Ltd, 2016)
Tang, Jonathan CY
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The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001
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Potent Delivery of Functional Proteins into Mammalian Cells in Vitro and in Vivo Using a Supercharged Protein
(American Chemical Society (ACS), 2010)
Cronican, James J.
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Thompson, David B.
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Beier, Kevin T.
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McNaughton, Brian R.
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The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics. When fused to superpositively charged GFP, proteins rapidly (within minutes) entered five different types of mammalian cells with potency up to ∼100-fold greater than that of corresponding fusions with known protein transduction domains (PTDs) including Tat, oligoarginine, and penetratin. Ubiquitin-fused supercharged GFP when incubated with human cells was partially deubiquitinated, suggesting that proteins delivered with supercharged GFP can access the cytosol. Likewise, supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro and also delivered functional recombinase enzyme to the retinae of mice when injected in vivo.
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A Hybrid Photoreceptor Expressing Both Rod and Cone Genes in a Mouse Model of Enhanced S-Cone Syndrome
(Public Library of Science, 2005)
Corbo, Joseph C
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Rod and cone photoreceptors subserve vision under dim and bright light conditions, respectively. The differences in their function are thought to stem from their different gene expression patterns, morphologies, and synaptic connectivities. In this study, we have examined the photoreceptor cells of the retinal degeneration 7 (rd7) mutant mouse, a model for the human enhanced S-cone syndrome (ESCS). This mutant carries a spontaneous deletion in the mouse ortholog of NR2E3, an orphan nuclear receptor transcription factor mutated in ESCS. Employing microarray and in situ hybridization analysis we have found that the rd7 retina contains a modestly increased number of S-opsin–expressing cells that ultrastructurally appear to be normal cones. Strikingly, the majority of the photoreceptors in the rd7 retina represent a morphologically hybrid cell type that expresses both rod- and cone-specific genes. In addition, in situ hybridization screening of genes shown to be up-regulated in the rd7 mutant retina by microarray identified ten new cone-specific or cone-enriched genes with a wide range of biochemical functions, including two genes specifically involved in glucose/glycogen metabolism. We suggest that the abnormal electroretinograms, slow retinal degeneration, and retinal dysmorphology seen in humans with ESCS may, in part, be attributable to the aberrant function of a hybrid photoreceptor cell type similar to that identified in this study. The functional diversity of the novel cone-specific genes identified here indicates molecular differences between rods and cones extending far beyond those previously discovered.
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Gene Expression Changes within Müller Glial Cells in Retinitis Pigmentosa
(Molecular Vision, 2012)
Roesch, Karin
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Stadler, Michael B.
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Purpose: Retinitis pigmentosa (RP) is a progressive retinal degeneration in which the retina loses nearly all of its photoreceptor cells and undergoes major structural changes. Little is known regarding the role the resident glia, the Müller glia, play in the progression of the disease. In this article, we define gene expression changes in Müller glial cells (MGCs) from two different mouse models of RP, the retinal degeneration 1 (rd1) and rhodopsin knockout (Rhod-ko) models. The RNA repertoire of single MGCs was comprehensively profiled, and a comparison was made between MGCs from wild-type (WT) and mutant retinas. Two time points were chosen for analysis, one at the peak of rod photoreceptor death and one during the period of cone photoreceptor death. Methods: Retinas were dissociated, and single MGCs were chosen under a dissecting microscope using a micropipette. Single cell cDNAs were generated and genome-wide profiles were obtained by hybridization to Affymetrix arrays. A comparison was made among all samples to discover the changes in gene expression during the periods of rod and cone photoreceptor death. Results: MGCs respond to retinal degeneration by undergoing gliosis, a process marked by the upregulation of glial fibrillary acidic protein (Gfap). Many additional transcripts were found to change. These can be placed into functional clusters, such as retinal remodeling, stress response, and immune-related response. Conclusions: A high degree of heterogeneity among the individual cells was observed, possibly due to their different spatial proximities to dying cells and/or inherent heterogeneity among MGCs.
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Clustering Analysis of SAGE Data using a Poisson Approach
(BioMed Central, 2004)
Cai, Li
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Huang, Haiyan
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Blackshaw, Seth
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Serial analysis of gene expression (SAGE) data have been poorly exploited by clustering analysis owing to the lack of appropriate statistical methods that consider their specific properties. We modeled SAGE data by Poisson statistics and developed two Poisson-based distances. Their application to simulated and experimental mouse retina data show that the Poisson-based distances are more appropriate and reliable for analyzing SAGE data compared to other commonly used distances or similarity measures such as Pearson correlation or Euclidean distance.
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Neuroanatomy goes viral!
(Frontiers Media S.A., 2015)
Nassi, Jonathan J.
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Beier, Kevin T.
The nervous system is complex not simply because of the enormous number of neurons it contains but by virtue of the specificity with which they are connected. Unraveling this specificity is the task of neuroanatomy. In this endeavor, neuroanatomists have traditionally exploited an impressive array of tools ranging from the Golgi method to electron microscopy. An ideal method for studying anatomy would label neurons that are interconnected, and, in addition, allow expression of foreign genes in these neurons. Fortuitously, nature has already partially developed such a method in the form of neurotropic viruses, which have evolved to deliver their genetic material between synaptically connected neurons while largely eluding glia and the immune system. While these characteristics make some of these viruses a threat to human health, simple modifications allow them to be used in controlled experimental settings, thus enabling neuroanatomists to trace multi-synaptic connections within and across brain regions. Wild-type neurotropic viruses, such as rabies and alpha-herpes virus, have already contributed greatly to our understanding of brain connectivity, and modern molecular techniques have enabled the construction of recombinant forms of these and other viruses. These newly engineered reagents are particularly useful, as they can target genetically defined populations of neurons, spread only one synapse to either inputs or outputs, and carry instructions by which the targeted neurons can be made to express exogenous proteins, such as calcium sensors or light-sensitive ion channels, that can be used to study neuronal function. In this review, we address these uniquely powerful features of the viruses already in the neuroanatomist’s toolbox, as well as the aspects of their biology that currently limit their utility. Based on the latter, we consider strategies for improving viral tracing methods by reducing toxicity, improving control of transsynaptic spread, and extending the range of species that can be studied.
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Preferential Budding of Vesicular Stomatitis Virus from the Basolateral Surface of Polarized Epithelial Cells Is Not Solely Directed by Matrix Protein or Glycoprotein
(American Society for Microbiology, 2015)
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Soh, Timothy K.
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Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.
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Haploinsufficient Bmp4 Ocular Phenotypes Include Anterior Segment Dysgenesis with Elevated Intraocular Pressure
(BioMed Central, 2001)
Chang, Bo
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Peters, Maureen
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Zabaleta, Adriana
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Nusinowitz, Steven
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Smith, Richard S.
Background: Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. Results: \(Bmp4^{+/-}\) mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. Conclusions: We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. \(Bmp4^{+/-}\) mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.
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RCAS-RNAi: A Loss-of-Function Method for the Developing Chick Retina
(BioMed Central, 2006)
Harpavat, Sanjiv
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Background: The embryonic chick provides an excellent model system for studies of development. However, it has lacked an efficient loss-of-function method for studies of gene function. Results: We show that avian retroviruses can deliver hairpins mediating RNA interference to the developing chick eye. These viruses 'knock down' specific genes in infected areas of the retina. The knock down persists as the retina matures and can be detected using in situ hybridization. Furthermore, the amount of retinal tissue affected can be controlled by manipulating the degree of infection. Conclusion: This technique provides a rapid and efficient loss-of-function option for studies in the developing chick retina.
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Ultrasound-Guided In Utero Injections Allow Studies of the Development and Function of the Eye
(Wiley-Liss, Inc., 2008)
Punzo, Claudio
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Ultrasound-guided in utero injections into the brain of murine embryos has been shown to facilitate gene delivery. We investigated whether these methods would allow gene transfer into ocular structures. Gene transfer using retroviral vectors or electroporation was found to be quite effective. We determined the window of time, as well as compared several strains of mice, that yield a high degree of survival and successful gene transfer. Several retroviral constructs were tested for expression and coexpresssion of two genes in retinal cell types. In addition, a retroviral vector was engineered to give cone photoreceptor-enriched expression, and a retroviral vector was demonstrated to provide RNAi-mediated loss-of-function. These methods enable access to early ocular structures and provide a more rapid method of assessment of gene and promoter function than possible using genetically engineered mice.

Cepko, Constance

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