Services

VIRUS SERVICES

The aim of the Core is to provide well characterized third generation lentiviruses, encoding cPPT and human hepatitis PRE, that have been titered and monitored for replication competent virus (RCR). We will provide services and advice on the use of lentiviruses to DERC investigators and will consult with investigators in the generation and preparation of lentiviruses they require. We have constructed a third generation lentivirus expression plasmid, pRRL-cPPT-CMV-X-PRE-SIN, that incorporates a central polypurine tract (cPPT) and a posttranscriptional element from human hepatitis B virus (PRE). The Core will generate lentiviruses encoding promoters to achieve tissue specific transgene expression, for example vectors encoding a liver specific promoter. We believe our two step centrifugation protocol produces virus preparations that are not toxic to cells in vitro or in vivo.

Murine retrovirus vectors provide highly efficient gene transfer and are particularly useful for ex-vivo gene transfer. Recombinant lentiviruses do not encode viral proteins and have the benefit of low toxicity and immunogenicity, produce stable proviral integrants and are the vector of choice for in vivo gene transfer. For users lacking expertise in molecular biology the core will clone a desired gene into an expression plasmid under the control of a promoter specified by the investigator. Genes can be obtained from published sources or can be cloned from mRNA isolated from specific tissues. We plan that the Core will have the expertise to generate high titer purified lentivirus encoding genes and promoters specified by investigators. The Core will provide consultation to investigators planning to use lentivirus virus vectors in their research.

The Core produces three virus systems: A. Lentivirus; B. Murine retrovirus; and C. Adenovirus. The Core will offer advice and training in the use of these vectors. Lentivirus vectors are especially useful for the transduction of non-proliferating cells, are not immunogenic and are prepared free of replication competent virus. The Virus Core will offer the service of cloning promoters and cDNA’s into expression plasmids for users.

A. Lentivirus vectors

The Core will produce well characterized lentivirus vectors encoding cPPT and human hepatitis PRE. Lentiviral vectors have the advantage over murine leukemia viral vectors of enabling provirus integration into non-dividing cells. These vectors are constructed by incorporating elements from human immunodeficiency virus 1(HIV1) that interact with the nuclear import system and mediate transport via the nucleopore into the cell’s nucleus. The Core will generate expression plasmids encoding genes and promoters specified by users. The Core will generate lentiviral vectors with tissue-specific promoters, lentiviral vectors with reporter genes and lentivirus based RNAi vectors. Reporter genes such as GFP or beta galactosidase would be coordinately expressed using IRES derived from EMCV. Lentiviral vectors will be pseudotyped with envelope glycoproteins from amphotropic murine leukemia virus (MLV) or vesicular stomatitis virus G protein (VSV-G). Two major benefits conferred by VSV-G pseudotyping are a broad tropism and a more robust virus that can be easily concentrated by centrifugation.

B. Murine retrovirus vectors

The Core will generate and titer murine retrovirus vectors (MMLV) from PA317 or GP13 packaging cells. Expression plasmids would encode the gene of interest and a selectable marker gene or a reporter gene. Other packaging cells will be used if requested.

C. Adenovirus Vectors

The Core plans to use an adenovirus vector system employing a backbone plasmid (pAdEasy) and a shuttle vector encoding the gene of interest. These vectors can be made with beta galactosidase or GFP reporter genes. Adenovirus will be titered and purified.

MOLECULAR BIOLOGY SERVICES

The Core offers quantitative real time RT-PCR, single nucleotide polymorphism (SNP) or microsatellite genotyping, purification and quantification of DNA and RNA from tissues, cloning genes into plasmid vectors and carrying out sequencing reactions. The Core serves the Affiliates of the Diabetes and Endocrinology Research Center in the area of Molecular Biology in relation to diabetes and endocrinology research. The Core develops new techniques, provides training and education, and assists in project design. Services include:

• Quantitative PCR - provides quantitative, or real-time PCR using a Stratagene Mx4000 quantitative PCR instrument. The Core also designs probes - TaqMan and MGB (minor groove binding) and primers for your gene of interest, and provides at no charge several probes for normalizing genes. Three genes in the same reaction can be measured.

• RNA isolation - of total or poly A+ RNA from tissue cultured cells, tissues, or blood.

• DNA purification - genomic DNA from rat, mouse, or human tissues, or plasmid DNA.

• Cloning - cloning genes from cDNA into plasmid vectors, site-directed mutagenesis.

• Sequencing - carry out DNA sequence reactions and obtain sequences.

• Northern blotting - prepare blot, hybridize with probe, image the results, and quantitate.

• RNA or DNA from Laser capture microdisection, and RNAi

• Consulting - assistance is provided in project design and analysis of results.

CELL SERVICES

The purpose of the Core is to stimulate and support the use of tissue culture and isolated tissues and to assist with the performance of viral transduction studies by Affiliate Investigators, especially those whose laboratories are not set up for tissue culture experiments. Experimental protocols using cell culture to study cell biology and gene expression are accessible to Affiliate Investigators who are not familiar or expert in these techniques. Isolation and procurement of human and non-human primate tissues is also supported. Highly specialized services are provided at uniform high yield and quality.

The Core services include:

• Providing access to tissue culture-related equipment.

• Preparation of culture medium, balanced salt solutions, and buffers.

• Providing cells and cell cultures from a central repository, expansion of primary cultures to include hepatocytes and adipocytes.

• Preparation of buffy coats and DNA.

• Expansion of proteomic studies from cultured cells.

• Production of tissue for laser capture microdissection (LCM).

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