Catalog - Vector Development Laboratory

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.

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 inital 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 serive is also used when investigators want an adenovirus expanded on the large-scale but do not have the 1x10^12 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 adenvirus. 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 1x10^12 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.

AAV vectors are produced using a dual transfection system. The first plasmid (pZAC2.1) contains an expression cassette (CMV promoter, multiple cloning site and SV40 polyA) flanked by AAV2 ITRs. The second plasmid contains the AAV2 rep and cap genes and adenovirus helper genes (E2, E4, VA1/II). Fifty 150 mm dishes of 293 cells are transfected. Virus is harvested and purified via ultracentrifugation banding on a cesium chloride gradient. The virus dialyzed, diluted and stored in a specific storage buffer.
To initiate service, contact us and we will send you an aliquot of pZAC2.1. Once you have cloned your gene of interest into pZAC2.1, send us 500 ug of the purified plasmid

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)
A new cell line producing a retroviral vector is created. The cell lines are created by transfection of a retroviral vector into a packaging cell line supplying virion gene products. Cell clones are selected and the investigator receives twenty such frozen clones.

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.

Lentivirus Services

Creation of Lentiviral Vector by Transiet 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.

Non-Viral Services

Plasmids

Laboratory-Scale Production
Bacterial cultures are grown in a 7.5 liter fermenter. After 15-24 hours the cells are collected, lysed, filtered and tranferred to a concentrator where the volume is reduced by 1/10-1/15 of the original volume. Plasmid isolation and purification is performed using a 3 column FPLC protocol that removes RNA, buffers, endotoxin and other contaminants and concentrates the plasmids. Several quality control assays are performed on the DNA to ensure its identity, sterility, and removal of endotoxins. Laboratory-scale plasmid production yeild 40-80 mg of plasmid.

Pilot-Scale Production
Bacterial cultures are grown in a 20 liter fermenter. After 15-24 hours the cells are collected, lysed, filtered and tranferred to a concentrator where the volume is reduced by 1/10-1/15 of the original volume. Plasmid isolation and purification is performed using a 3 column FPLC protocol that removes RNA, buffers, endotoxin and other contaminants and concentrates the plasmids. Several quality control assays are performed on the DNA to ensure its identity, sterility, and removal of endotoxins. Laboratory-scale plasmid production yeild ~200 mg of plasmid.

Lipsomes

Liposomes
The VDL supplies bulk manufactured liposomes directly for use in tissue culture or animal experiments. We will provide critical QA/QC information about these liposome complexes by performing sub-micron particle size analysis, and by determining overall charge by zeta potential analysis.

Nucleic acid:Liposome complexes - non-targeted
The VDL will prepare nucleic acid:liposome complexes using our liposomes and plasmid preparations provided by the investigator. We will provide critical QA/QC information about these liposome complexes by performing sub-micron particle size analysis, and by determining overall charge by zeta potential analysis.

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 overlayed 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. Invidual 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 valiadation and will be available soon.

AAV

Determine number of genome equivalents
The concentration of genome equivalents of recombinant AAV is measured by quantitative real time 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 infectious unit concentration
Recombinant AAV infectivity is determined by infection of 293 cells with rAAV and with or without wildtype AAV and wildtype Adenovirus. Infectious units are determined by the transfer of cells infected with dilutions of the rAAV to membrane filters followed by hybridization with a probe corresponding to the rAAV sequence and counting of labeled infectious centers.

Assay for replication competent AAV
The content of replication competent AAV is also determined using quantitative real time PCR, except the primer/probe combination is within the cap sequence and the standard curve is prepared using wild type AAV DNA containing the cap sequence.

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.

Identity Testing
Identify testing is performed using restriction digest of the test sample and comparing the molecular weight of the restriction fragments to the predicted restriction pattern.

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 specturm which results in much lower background levels than green/yellow emitting luciferases.

Ad5-Halo tag -

Ad5-dsRed -

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 does not 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. 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.

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



	Catalog & Ordering / Catalog / Ordering 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 inital 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 serive is also used when investigators want an adenovirus expanded on the large-scale but do not have the 1x10^12 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 adenvirus. 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 1x10^12 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. Adeno-Associated Virus Services Creation and Purification AAV vectors are produced using a dual transfection system. The first plasmid (pZAC2.1) contains an expression cassette (CMV promoter, multiple cloning site and SV40 polyA) flanked by AAV2 ITRs. The second plasmid contains the AAV2 rep and cap genes and adenovirus helper genes (E2, E4, VA1/II). Fifty 150 mm dishes of 293 cells are transfected. Virus is harvested and purified via ultracentrifugation banding on a cesium chloride gradient. The virus dialyzed, diluted and stored in a specific storage buffer. To initiate service, contact us and we will send you an aliquot of pZAC2.1. Once you have cloned your gene of interest into pZAC2.1, send us 500 ug of the purified plasmid 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) A new cell line producing a retroviral vector is created. The cell lines are created by transfection of a retroviral vector into a packaging cell line supplying virion gene products. Cell clones are selected and the investigator receives twenty such frozen clones. 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. Lentivirus Services Creation of Lentiviral Vector by Transiet 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. Non-Viral Services Plasmids Laboratory-Scale Production Bacterial cultures are grown in a 7.5 liter fermenter. After 15-24 hours the cells are collected, lysed, filtered and tranferred to a concentrator where the volume is reduced by 1/10-1/15 of the original volume. Plasmid isolation and purification is performed using a 3 column FPLC protocol that removes RNA, buffers, endotoxin and other contaminants and concentrates the plasmids. Several quality control assays are performed on the DNA to ensure its identity, sterility, and removal of endotoxins. Laboratory-scale plasmid production yeild 40-80 mg of plasmid. Pilot-Scale Production Bacterial cultures are grown in a 20 liter fermenter. After 15-24 hours the cells are collected, lysed, filtered and tranferred to a concentrator where the volume is reduced by 1/10-1/15 of the original volume. Plasmid isolation and purification is performed using a 3 column FPLC protocol that removes RNA, buffers, endotoxin and other contaminants and concentrates the plasmids. Several quality control assays are performed on the DNA to ensure its identity, sterility, and removal of endotoxins. Laboratory-scale plasmid production yeild ~200 mg of plasmid. Lipsomes Liposomes The VDL supplies bulk manufactured liposomes directly for use in tissue culture or animal experiments. We will provide critical QA/QC information about these liposome complexes by performing sub-micron particle size analysis, and by determining overall charge by zeta potential analysis. Nucleic acid:Liposome complexes - non-targeted The VDL will prepare nucleic acid:liposome complexes using our liposomes and plasmid preparations provided by the investigator. We will provide critical QA/QC information about these liposome complexes by performing sub-micron particle size analysis, and by determining overall charge by zeta potential analysis. 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 overlayed 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. Invidual 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 valiadation and will be available soon. AAV Determine number of genome equivalents The concentration of genome equivalents of recombinant AAV is measured by quantitative real time 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 infectious unit concentration Recombinant AAV infectivity is determined by infection of 293 cells with rAAV and with or without wildtype AAV and wildtype Adenovirus. Infectious units are determined by the transfer of cells infected with dilutions of the rAAV to membrane filters followed by hybridization with a probe corresponding to the rAAV sequence and counting of labeled infectious centers. Assay for replication competent AAV The content of replication competent AAV is also determined using quantitative real time PCR, except the primer/probe combination is within the cap sequence and the standard curve is prepared using wild type AAV DNA containing the cap sequence. 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. Identity Testing Identify testing is performed using restriction digest of the test sample and comparing the molecular weight of the restriction fragments to the predicted restriction pattern. 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 specturm which results in much lower background levels than green/yellow emitting luciferases. Ad5-Halo tag - Ad5-dsRed - 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 does not 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. 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. To request an initiate service, fill out the Service Request form and return it to us by fax (713-798-1230) or by e-mail (vector@bcm.edu).
	CAGT Home \| BCM Home \| Privacy Notices ©2002, CAGT Vector Development Laboratory, Baylor College of Medicine One Baylor Plaza, N1010, Houston, TX 77030, (713)798-1238 URL: http://vector.bcm.tmc.edu Email:vector@bcm.tmc.edu (Modified: April 16, 2007 FC)

Vector Development Lab Catalog