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Vector Development Laboratory

Houston, Texas

Vector Development Laboratory
CAGT Vector Development Laboratory, Baylor College of Medicine
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Vector Development Lab Catalog

Adenovirus Service

Creation of Ad5 vector

With this service, you will receive the pShuttleX transfer vector to clone your transgene. We ask that you send us 100 ug of the shuttle vector containing your transgene. Once we receive the shuttle vector, we will move your transgene into the pAdenoX (Clontech) and prepare the new construct for transfection. Following transfection, 6-8 discrete plaques will be selected and undergo small expansion. One-third of the small expansions will be sent to you for testing transgene expression. One-third of the small expansions will be used to extract and analyze viral DNAs. The final one-third will be stored in the -80 until you inform us of which isolate to expand.

Creation of Ad5F35 vector

With this service, you will receive the pShuttleX transfer vector to clone your transgene. We ask that you send us 100 ug of the shuttle vector containing your transgene. Once we receive the shuttle vector, we will move your transgene into the pAd5F35 vector and prepare the new construct for transfection. Following transfection, 6-8 discrete plaques will be selected and undergo small expansion. One-third of the small expansions will be sent to you for testing transgene expression. One-third of the small expansions will be used to extract and analyze viral DNAs. The final one-third will be placed in storage until you inform us of which isolate to expand.

Transfection and plaque isolation of Ad vector

With this service, you do all the cloning. Once you have inserted your transgene into one of the Ad plasmid vectors, you send us 100 ug of the plasmid and we prepare your construct for transfection. Following transfection, 6-8 discrete plaques will be selected and undergo small expansion. One-third of the small expansions will be sent to you for testing transgene expression. One-third of the small expansions will be used to extract and analyze viral DNAs. The final one-third will be stored in the -80 until you inform us of which isolate to expand.

Small-scale adenovirus expansion

We have found some investigators wishing to expand an Ad vector which they obtained from a second party often have minimal information about the vector titer, purification state, age, freeze/thaw history, etc. This precludes us from beginning with the large-scale expansion. To circumvent this problem, we begin by infecting one 60mm dishes of 293 cells with the virus. We collect virus and use it to do a medium-scale infection (a.k.a. preparation of lysate).

Preparation of lysate (a.k.a. medium-scale expansion)

This service is most commonly used when we have created a custom virus. Crude virus from the initial plaque expansion is used to infect 5, 150 mm dishes of 293 cells. The lysate from this expansion is used to infected the cells for the large-scale expansion. This service is also used when investigators want an adenovirus expanded on the large-scale but do not have the 1x1012 particles needed for infection. We infect 5, 150 mm dishes of 293 cells with ~2500 particles/cell. The crude virus produced is used in the large-scale expansion.

Large-scale adenovirus expansion and purification

The large-scale expansion is the final expansion in the production of an adenovirus. The infection begins with either 1x10^12 particles of purified virus or crude viral lysate collected from the medium-scale expansion. If you do not have 1x1012 particles of purified virus see "Preparation of lysate" above. The crude virus collected from the cell factory is purified using cesium chloride ultracentrifugation. The purified virus is desalted and diluted in a specific storage buffer to 5x10^12 particles/ml.

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Retrovirus Services

Retrovirus (Transient Transfection) - Culture Supernatant

A packaging cell line based upon 293T cells is transfected with the investigator's retroviral construct and supernatant (4 ml) is collected and supplied to the investigator.

New Retroviral Producer (Transfection & Infection)

A new cell line producing a retroviral vector is created. The cell lines are created by transfection of a retroviral vector into either an amphotropic or ecotropic packaging cell line supplying virion gene products. Culture supernatant from this transfection is used to then infect a packaging cell line supplying an envelope protein of host range that is different from the original packaging cell line (e.g. an amphotropic packaging cell line is infected if an ecotropic packaging cell line was first transfected). This cross-infection procedure generates a more homogenous population of clones for screening and usually results in producer cell lines yielding higher titers of vector than transfection alone. The investigator receives 6-8 frozen clones.

Retrovirus Screened Producer (10 Clones)

The cell clones from the process of creating a new retroviral producer are screened by infection of a permissive cell line followed either by assay for the expressed protein or by DNA extraction and analysis by Southern blot.

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Lentivirus Services

Creation of Lentiviral Vector by Transient Transfection

The first step in lentivirus creation is to be completed by the client. The VDL will provide you will a transfer plasmid for cloning your transgene. Once you have cloned your gene, return 50 ug of the plasmid to us for recombination into one of the pLenti expression plasmids using Gateway Technology. We will grow a large-scale prep of the plasmid for co-tranfection with the ViraPower Packing Mix into 293FT producer cell line. At 48-72 hours post-transfection, the viral supernatant is harvested.

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Quality Control/Quality Assurance Services

Adenovirus

Plaque Assay - to determine infectivity titer

The standard protocol involves making 6 serial dilutions of the virus and infecting 2, 6-well plates of 293 cells with the virus. At ~16 hours post-infection, the cultures are overlaid with an agar/media mixture. The cells are observed daily for the formation of plaques. The plaques are counted and the applied to a formula that will give the infectious titer/ml. The protocol takes 10-14 days to complete.

Rapid Titer Assay - to determination of infectivity titer

We use the AdenoX Rapid Titer kit (Clontech) for this service. This immunohistochemical assay involves making 3 serials dilutions of the virus and infecting 6 wells of a 12 well plate of 293 cells. At 48 hours post-infection, the cells are fixed and immunostained with an anti-hexon antibody. Individual or clusters of stained cells are counted and the resulting figures are applied to a formula that will give th infectious titer/ml. This protocol takes 3 days to complete.

Assay for Replication Competent Adenovirus (RCA) - Cell-Based Assay

Preparations are judged to be free of replication competent adenovirus if they pass a test of incubation of 10^9 particles of recombinant Ad on A549 cells for 10 days. Appropriate positive and negative controls are run with each RCA test.

Assay for Replication Competent Adenovirus (RCA) - Quantitative Real-Time PCR

A new rapid RCA test using real time quantitative PCR using primer/probes within Ad region E1 has been developed. This assay is currently undergoing the final steps of validation and will be available soon.

Retrovirus

Quantify concentration of genome equivalents

The concentration of retrovirus genome equivalents is measured by quantitative real time reverse transcription-PCR (ABI Taqman) using primers/probes (FAM-non-fluorescent) within the SV40 polyadenylation sequence. Standard curves are prepared using plasmids containing the same sequence.


Determine infectivity titer

The infectivity titer of retroviral vectors is highly dependent upon the transgene cassette. In cases where a selectable marker is present within the vector (e.g. neomycin) titration is a matter of plating transduced cells in the presence of the agent (e.g. G418). Also, if the transgene is easily measured (e.g. GFP), it can also be used as a means of vector titration.

Assay for Replication Competent Retrovirus

The presence of replication competent retrovirus in retroviral stock is checked by marker rescue assay. Cells not expressing retroviral proteins have been infected with a retrovirus expressing the marker green fluorescent protein. A cell line has been selected. This cell line is infected with the retrovirus stock in question and the supernatant harvested and tested on naive cells. Any green fluorescent protein expression on these cells indicates the presence of replication competent retrovirus in the stock.

Lentivirus

Quantify concentration of genome equivalents

Determining the particle titer is performed by extraction of viral RNA from the lentiviral vector followed by titration by quantitative real time RT-PCR (ABI, Taqman) using primer/probes within the LTR. A standard curve is generated by using T7 transcripts of sequences containing the same LTR.

Determine the infectivity titer

As with all retroviral vectors, determining the infectivity titer of lentiviral vectors is dependent upon the expression cassette. In cases where a selectable marker is present within the vector (e.g. neomycin) titration is a matter of plating transduced cells in the presence of the agent (e.g. G418). Also, if the transgene is easily measured (e.g. GFP), it can also be used as a means of vector titration.

All Vectors

Test for sterility

Sterility testing is performed using Millipore Steritest kits containing Trypticase Soy Broth for detection of yeast, mold, and aerobic bacteria and fluid thioglycollate medium for detection of yeast, mold, aerobic and facultative anaerobic bacteria.

Test for endotoxin contamination

Endotoxin testing is performed using a quantitative kinetic-chromogenic limulus amebocyte lysate or LAL test (USP 28: 2005)

Test for mycoplasma contamination

Cell substrates for vector production (e.g. Phoenix A, 293, 293 T) are routinely tested for mycoplasma (MycoAlert®, Cambrex).

Test for host cell DNA contamination

DNA contamination is detected by real time quantitative PCR for DNA encoding 18S ribosomal RNA with a standard curve against cloned 18S ribosomal DNA.

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In Stock Adenoviruses

Ad5-CMV-GFP

The Ad5-CMV-GFP virus is a powerful tool for determining the transfection efficiency of an Ad5 virus in your cells of interest. Examining cells for GFP expression following transduction with Ad5-CMV-GFP using a fluorescent microscope is a quick method for estimating transduction efficiency.

Ad5-CMV-lacZ

The Ad5-CMV-lacZ virus is a powerful tool for determining the transfection efficiency of an Ad5 virus in your cells of interest. Examining cells for beta-galactisidase activity following transduction with Ad5-CMV-lacZ is an easy method for estimating transduction efficiency.

Ad5-CMV-TK

The Ad5-CMV-TK virus expresses the thymidine kinase gene.

Ad5-CMV-BFP

The Ad5-CMV-BFP virus expresses the blue florescent protein.

Ad5-CMV-Cre

The Ad5-CMV-Cre expresses the cre recombinase protein.

Ad5-CMV-Cre-GFP

The Ad5-CMV-Cre-GFP expresses the cre recombinase protein along with GFP. The transcription of GFP is driven by the IRES so the GFP expression will be slightly less than seen in the Ad5-CMV-GFP vector.

Ad5-CMV-CBR-luciferase

This adenovirus expresses the Click Beetle Red (CBR)luciferase gene developed by Promega. The Ad5-CMV-CBR-luciferase is an excellent tool for live in vivo tracking of the adenovirus migration. The thing that makes this tagged vector better than others is that it emits in the red range of the spectrum which results in much lower background levels than green/yellow emitting luciferases.

Ad5-CMV-Halo tag

This recombinant human adenovirus type 5 expresses the HaloTag protein under the control of CMV promoter. Ad5-CMV-HaloTag serves as a control for other recombinant adenoviruses.

Ad5-Empty

The Ad5-Empty virus does not contain a promoter, transgene or poly(A). This virus is often used as a control virus in in vitro and in vivo studies.

Ad5-CMV-Empty

The Ad5-Empty virus contains a CMV promoter and poly(A) but no transgene.. This virus is often used as a control virus in in vitro and in vivo studies.

Ad5F35-CMV-GFP

The Ad5F35-CMV-GFP virus is a powerful tool for determining the transfection efficiency of an Ad5F35 virus in your cells of interest. Examining cells for GFP expression following transduction with Ad5F35-CMV-GFP using a fluorescent microscope is a quick method for estimating transduction efficiency. Learn more about the Ad5F35 viruses.

Ad5F35-Empty

The Ad5F35-Empty virus does not contain a promoter, transgene or poly(A). This virus is often used as a control virus in in vitro and in vivo studies. Learn more about the Ad5F35 viruses.

Ad5-CMV-dsRed

This recombinant human adenovirus type 5 expresses Discosoma sp. red fluorescent protein (DsRed) under the control of CMV promoter. Ad5-CMV-dsRed serves as a control for other recombinant adenoviruses. Examining cells for dsRed expression following transduction with Ad5-CMV-dsRed using a fluorescent microscope is a quick method for estimating transduction efficiency.

In Stock Retroviruses

Retro-GFP

The Retro-GFP

Retro-dsRed

The Retro-dsRed

In Stock Lentiviruses

Lenti-GFP

The Lenti-GFP

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To request an initiate service, fill out the Service Request Form and e-mail it to vector@bcm.edu or return it to us by fax at 713-798-1230.

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