GENE EXPRESSION VECTOR CORE

Aims:

• To support the development of HSV-based amplicon vectors for in vitro and in vivo gene transfer for aging-related studies.
• To support rapid quantitative measurements of DNA and RNA using real-time PCR for aging-related studies.

Introduction:

The facilitation of molecular genetic methods to the study of mechanism(s) of aging-related diseases and the development of novel gene delivery-based therapies is the primary focus of the Gene Expression Vector Core (GEVC). The core has demonstrated success in the design of gene delivery vectors based upon the Herpes Simplex Virus (HSV)-derived amplicon, as well as, expertise in ultrasensitive and quantitative methods for nucleic acids. The plasmid-based amplicon is a highly versatile platform, owing to its facility to molecular genetic manipulation. The amplicon is essentially a eukaryotic expression plasmid modified by the addition of a HSV origin of DNA replication (ori) and cleavage/packaging sequence ("a" sequence). Heterologous transcription units of various size (theoretical limit is 150 kb minus the size of the parental amplicon) can be cloned into an amplicon plasmid and then packaged into virions. Amplicons have been utilized experimentally to transfer a variety of genes to many cell types, but their potential usefulness in aging research remains to be exploited. Similarly, high throughput, quantitative methods for detection of specific RNA or DNA sequences in cells or tissues have not been widely used because of the related high equipment and reagent costs.

Facilities and Services:

Function I. Generation of virus vectors for gene transfer into primary neural cells and the CNS Gene delivery and expression (transduction) into primary cell cultures and in vivo is most efficient using virus vectors. The Core has specialized expertise in the generation of non-cytopathic herpes simplex virus amplicon vectors. These plasmid-based vectors are easily constructed and allow the investigator to choose which promoter and gene to express (Table 1). The amplicon cloning vectors do not contain any viral genes. The amplicon cloning vectors are converted into virus using the helper virus-free BAC method. This process requires four days for virus generation, two days for purification and concentration, and an additional two days for titer determination. Using Core developed second generation BAC and complementing cell lines no recombinant virus is detected. Thus, the only virions produced are those which contain a virus genome consisting of 20 to 30 copies of the gene of interest. Titering of virus is provided to the investigator for either tissue culture or in vivo use.

Following an initial consultation with the Core director and staff, the investigator will be provided with the appropriate amplicon cloning vector plasmids and plasmid maps that best suit the desired application. The amplicon cloning vectors that are available are shown in Table 1. It will be the investigator's responsibility to then clone the DNA fragments of interest into the selected vector. The newly constructed amplicon plasmid will be returned to the Core, where it will be purified in large quantities and subsequently packaged into viral particles. The virus stocks will be titered according to the investigator's method of choice: either by expression units or by transduced genome equivalents. The assessment of expression units will require the use of a histochemical or immunocytochemical method. If an immunocytochemical method is required, it will be the investigator's responsibility to provide the appropriate specific antibody and protocols for efficient titer determination by Core technical staff. Real-time quantitative PCR will be performed by the Core to determine virus titers using the b-lactamase primer/probe combination shown in Table 2 if so desired by the investigator.

Function II. Quantitative nucleic acid measurements Specific target nucleic acids derived from cells, tissues, and bodily fluids need to be measured quantitatively, rapidly, and often repeatedly. The PCR satisfies the requirement for rapid and multiple measurements but suffers from lack of quantitative power when used conventionally. Real-time quantitative PCR circumvents the limitations of conventional PCR approaches by utilizing a technology that counts each cycle of product formation. The liberation of a fluorescent molecule is detected by fiber optic transduced device incorporated in the ABI Prism 7700. The quantitation system also allows for multiplexing of several primer/probe combinations through the utilization of different coupled fluorescent dyes to determine relative target DNA levels (Table 2). This instrument and its technical staff will be available to interested investigators. Consultation for probe sequence design and technical support for measurements are to be implemented.

Genomic DNA from archived or supplied cells, virus, and mRNA are converted to cDNA and then become useful targets for quantitative PCR. Investigators will prepare samples in their own laboratories using Core-supplied protocols. In consultation with the Core Director and staff optimal probe sequences will be identified and ordered. Pilot standard curves will be performed on known material to establish conditions for optimum cycle threshold (CT) determination. Once conditions are optimized, the Core will run all investigator samples in duplex with a rDNA standard if starting material is cell-derived. In the case of bodily fluids, reconstruction standard curves with known amounts of another target DNA will be run in parallel to ensure starting material and template integrity. All data will be transferred to Microsoft Excel on the Core Server for later retrieval and analyses by the investigator. Quantitative nucleic acid determinations will also be made for amplicon virus titering (see Core Function I).

Table 1: Amplicon Plasmid Vectors Available for Cloning

Table 2: PE770 Primer/Probe Combinations Presently Available

 

Set Name	Sense Primer	Antisense Primer	Probe	Coupled Dye
Ad2MLP (a promoter in a parental vector)	5'GGTCAAAAACCAGG TTTGGCTATA3'	5'TCCTGGCCCTC GCAGA3'	5'CGTCCTCACTCTC TTCCGCATCGC3'	TET
LacZ (a frequently used reporter gene)	5'GGGATCTGCCATTG TCAGACAT3'	5'TGGTGTGGGCC ATAATTCAA3'	5'ACCCCGTACGTC TTCCCGAGCG3'	FAM
18S rRNA (a normalization std)	5'CGGCTACCACATCC AAGGAA3'	5'GCTGGAATTACC GCGGCT3'	5'TGCTGGCACCAG ACTTGCCCTC3'	JOE
b-lactamase (a gene in all amplicons)	l5'CTGGATGGAGGCGG ATAAAGT3'	5'CGGCTCCAGATT TATCAGCAAT3'	5'CAGGACCACTTC TGCGCTCGGC3'	FAM
ICP0 (a gene in BAC and HSV viruses)	5'ATGTTTCCCGTCTGG TCCAC3'	5'CCCTGTCGCCTT ACGTGAA3'	5'CCCCGTCTCCAT GTCCAGGATGG3'	FAM