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Comprehensive NeuroAIDS Center

COMPREHENSIVE NEUROAIDS CENTER (cnac)

Basic Science Core I: Cell Culture and Neurotropic Viruses and Proteomics

 

This Core provides mammalian cell culture and virology services to investigators performing biomedical research on AIDS and the nervous system. Services include the preparation of CNS cell cultures, virus propagation, proteomics, biomarkers, and consultative expertise in gene editing. These services promote in uncovering mechanisms involved in the development of nervous system disorders associated with HIV-1 infection.

 

 

Basic Science Core I

Leadership

 

Kamel Khalili, PhD, Leader

T. Dianne Langford, PhD, Co-Leader

Salim Merali, PhD, Co-Leader

Wenhui Hu, MD, PhD, Co-Leader

 


Basic Science Core I

Mammalian Cell Culture and Neurotropic Viruses

 

This section of the Core focuses on the study of HIV-1 infection at the molecular and cellular levels. Services include the purification, characterization, and maintenance of an assortment of central nervous system cells (neurons, astrocytes, oligodendrocytes, microglia, cerebral endothelial cells, progenitor and neural stem cells) and peripheral cells such as macrophages and T-cells.

 

To request cell samples, please complete the Cell Request Form (PDF) and return to wyen@temple.edu.

 

For additional information, please contact Dianne Langford, PhD.

 

 Basic Science Core researchers identifying cells.  Photo by Ryan Brandenberg, Temple University.


 

Basic Science Core I

Gene Editing

 

Targeted genome editing has been explosively fueled by the introduction of several novel DNA cleavage (nuclease) technologies including homing endonucleases or meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR-associated system 9 (Cas9) nucleases1. These nucleases utilize site-specific double-strand DNA break (DSB)-mediated DNA repair mechanisms allowing precise genome editing. Such strategy has enabled rapid, easy and efficient modification of endogenous genes/genomes in a wide variety of cell types and species. Clinically, it is being explored as a novel therapeutic approach for genetic disorders, viral infections and cancer. All ZFN, TALEN and Cas9 techniques have been used to disrupt HIV-1 entry co-receptors (CCR5, CXCR4) and proviral DNA-encoding viral proteins2. CCR5 gene-targeting ZFNs are in phase 2 clinical trials for HIV-1/AIDS treatment3. Also, ZFN and Cas9/gRNA have been recently shown to remove the proviral HIV-1 DNA from host cellular genome, by targeting its highly-conserved 5’ and 3’ long term repeats (LTRs)4. Our recent studies found that LTR-directed Cas9 eradicates the HIV-1 genome and effectively vaccinates target cells against HIV-1 reactivation and infection with high specificity and efficiency5. These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses.


Traditional homologous recombination-mediated gene targeting has made a great breakthrough in transgenic animal models, but the low efficiency, time-consuming and high costs limit its routine application6. Site-specific DSBs allow precise and efficient genome editing1 with 100-50,000 fold increase in the targeting efficiency (Ellis et al., 2013). In the past three years, ZFN and TALEN have been widely used for such genome editing. However, both require the engineering of custom DNA-binding protein for each target site and both produce uncontrollable off-target effects7. In the past two years, the Cas9 biotechnology has dominated the field of genome editing because of its easy, fast, cheap and versatile features. Cas9, together with small guide RNA (sgRNA), generates DSBs at any site defined by a 20-nucleotide guide (seed) sequence and trinucleotide (NGG or NAG) protospacer adjacent motif (PAM) recognized by Cas98. This method needs only a tiny custom RNA molecule which, like small hairpin RNA (shRNA), can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6-promoted sgRNA expression vector. In addition to gene targeting, the Cas9/gRNA technology has been expanded to the gene regulation (activation and repression).


Cas9 and sgRNA can be expressed in individual vector or all-in-one vector. Various types of Cas9/gRNA expression vectors are available through Addgene or commercial companies. The Genome Editing Core at CNAC provides various types of consultations including experimental design, gRNA screening, Cas9/gRNA vector selection, genotyping analysis, specificity and off-target evaluation, and data interpretation. This Core also assists investigators with the generation of Cas9 stable cell lines and transgenic animals as well as target gene knockin/knockout cell lines and transgenic animals.

 

1 (Bedell et al., 2012; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013; Qi et al., 2013; Wang et al., 2012a)
2 (Manjunath et al., 2013; Stone et al., 2013)
3 (Hofer et al., 2013; Tebas et al., 2014)
4 (Ebina et al., 2013; Qu et al., 2013)
5 (Hu et al. 2014)
6 (Wang et al., 2012b)
7 (Bogdanove and Voytas, 2011; Chen et al., 2013; Lee et al., 2012; Mussolino et al., 2011)
8 (Gasiunas et al., 2012; Jinek et al., 2012)

 

 

Basic Science Core I

Proteomics

 

This Core provides state-of-the-art services in proteomics and data analysis, including novel methods for discovering biomarkers, innovative differential expression profiling and bioinformatics, interactomics (protein-protein interaction), and post-translational modifications (PTMs). In addition, this Core assists investigators with identification of discriminative proteins, protein function and pathway analysis, development of prognostic models, and modern methods for biomarker discovery from plasma and cerebral spinal fluid.

 

For additional information on proteomics, please contact Salim Merali, PhD.

 

Salim Merali, PhD

 

 

Basic Science Core I

News & Updates

 

The Comprehensive NeuroAIDS Center (CNAC) Basic Science Core I:


Gene editing strategy for eliminating HIV-1 DNA from cells.


This core now provides consultations on the use of gene editing strategy employing CRISPR/Cas9 for editing the gene of your interest in cell cultures and animal models. This service is provided by the newest member of the CNAC, Dr. Wenhui Hu, who can be contacted at wenhui.hu@temple.edu. More recently, this technology has been utilized by Dr. Khalili’s and Dr. Hu’s laboratories for eradicating the HIV-1 genome from the human cell cultures, as described below.


Highly active antiretroviral therapy (HAART) profoundly inhibits HIV-1 replication, yet has failed to eliminate the virus from infected individuals. Thus, there is an urgent need for the development of effective strategies that will lead to the eradication of HIV-1 infection from the human population and the cure of AIDS. The presence of the proviral DNA genome as integrated copies in the host chromosomes, persistent low level replication of HIV-1 in latently infected cells, and continued risk for resurgence of the virus in resting CD4+ memory T-cells from deep dormancy leave little doubt that removal of the viral genome from the host chromosome is the ultimate cure for AIDS. Recently, we have successfully employed newly developed gene editing techniques, based on RNA-guided Cas9, to specifically target the HIV-1 genome and eliminate integrated copies of the proviral DNA from latently infected cell lines. In this attempt, we identified several specific DNA sequences within the U3 region of the HIV-1 LTR that serve as targets and are efficiently edited by single and multiplex Cas9/gRNAs to completely abrogate viral replication in latently infected macrophages, T-cells, and microglial cells. Our Cas9/gRNAs inflict no genotoxicity or off-target editing to the host cells, yet precisely excise a 9709 bp DNA fragment of the integrated proviral genome that spans between the 5'- and 3'- LTR. Moreover, the presence of multiplex gRNAs and Cas9 in the cells protected cells against future or subsequent HIV-1 infection. These observations offer a strong rationale to hypothesize that this novel approach can serve as a platform to explore the permanent removal of HIV-1 from the host and a cure for AIDS.

 

CNAC Gene Editing Newsletter

 

Basic Science Core I

Posters

 

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COMPREHENSIVE NEUROAIDS CENTER BASIC SCIENCE CORE I: MAMMALIAN CELL AND NEUROTROPIC VIRUS FACILITY

 

 

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COMPREHENSIVE NEUROAIDS CENTER BASIC SCIENCE CORE I: PROTEOMICS AND METABOLOMICS FACILITY