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Bua application Details (BU041135-03)


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BUA Application Details (BU041135-03)



Summary

Principal Investigator :

McManus, Michael T.

PI Department :

Diabetes Center

Laboratory Supervisor :

Michael T. McManus

Title :

Lentiviral RNAi Core

Status :

Approved

Expiration date :

04/01/2018

OEH&S Specialist :

Herminigildo Briones

Application Type :

Continuation

NIH Level :

Section III-D-1



Study involves

Recombinant DNA materials or technology:

Yes

Infectious Agents (IA):

No

Bloodborne Pathogens (BBP):

Yes

Biological Toxins:

No

Select Agents :

No

Generate Genetically-Modified Animals:

No

Animals:

No

Shipping of biological materials:

Yes



Describe the goals of your research and an overview of the experimental design to attain these goals.

Among the 3 billion base pairs of human genome sequence, there are at least 30-40 thousand protein-encoding genes, but the function of at least half of them remains unknown. The use of lentiviral vectors and other viral agents (including MMLV, MSCV, and AAV) are powerful tools for systematically deciphering the functions and interactions of these thousands of genes. Their ability to transduce both dividing and non-dividing cell types allows for their application in both overexpression and RNAi experiments.

The overall goal of our research is to provide high titer virus to researchers in the Immunology and Microbiology Departments and the Diabetes Center and members of the UCSF community. This involves:

• Packaging of both shRNA, gRNA, and cDNA expressing self-inactivating lentiviral vectors, MMLV, AAV, and other viral agents upon request.

• Titration of produced virus by flow cytometry.

• Providing a resource for protocols and safety regarding work with infectious reagents.

All labs for which we will provide lentiviral or other vectors will have active BUAs that include these biomaterials.



No significant changes have occurred since our last BUA.



Application History

Project Number

Approval Type

Approval Date

Expiration Date

BU041135-03

Continuation

03/10/2015

04/01/2018

BU041135-02C

Modification

01/17/2014

03/01/2015

BU041135-02B

Modification

06/14/2013

03/01/2015

BU041135-02A

Modification

08/15/2012

03/01/2015

BU041135-02

Continuation

02/15/2012

03/01/2015

BU041135-01

New Approval




12/01/2011



Funding



Type:






Funding Agency/Sponsor

Grant or Contract Number



Applicant comments:

We are funded by recharge.



Associated Authorizations/Protocols



Regulated Materials

Approval Number

Legacy Number

Status

Expiration Date



Recombinant DNA Research



I am transferring genetic material into a microbiological vector or any organism in order to clone the gene, create a stable genetic alteration, obtain the expression product(s) of the gene(s), or silence/knock-down the target gene(s). The genetic material is not from the same species of organism or vector that is receiving it.

No

I am introducing foreign genetic material using a vectorless system (e.g. electroporation, lipofection).

Yes

The genetic material being cloned or expressed is from a Risk Group (RG) 3 agent.

No

The genetic material being cloned or expressed is from a Risk Group (RG) 4 agent.

No

The genetic material being cloned or expressed consists of more than two-thirds of the genome of a RG 1 or RG 2 Eukaryotic virus.

No

The genetic material being cloned or expressed encodes a known or reasonably suspected Oncogene or Teratogen.

No

The vector I am using for introduction of foreign genetic material into the host is, or is derived from, a RG 3 agent.

No

The vector I am using for introduction of foreign genetic material into the host is a RG 1 or RG 2 Eukaryotic virus which contains more than two-thirds of its viral genome, or more than two-thirds of another Eukaryotic virus' genome.

No

The vector I am using for introduction of foreign genetic material into the host is being used in conjunction with a helper virus.

No

The host into which I am introducing foreign genetic material is a cell or organism other than E. coli strain K12; S. cerevisiae, S. uvarum, B. Subtilis, or B. licheniformis?

Yes

I will be making more than 10 liters, in one container, of any culture.

No

I am using synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules and/or cells, organisms, and viruses containing such molecules.

No

The gene(s) I am transferring into a microorganism encode for antibiotic resistance that the organism would not acquire naturally, and the resistance could compromise the use of the antibiotic to control disease caused by the receiving organism in human medicine, veterinary medicine or agriculture.

No

The gene(s) I am transferring into a microorganism for biosynthesis of a toxin lethal to vertebrates with an LD50 of less than 100 nanograms per kilogram body weight (100ng/Kg).

No

I am transferring recombinant DNA, or DNA or RNA derived from recombinant DNA into one or more human research subjects.

No



Describe experimental procedures that will involve the rDNA listed here. You should tell the IBC (Institutional Biosafety Committee) not only what you will do but also how you will do it. Step-by-step descriptions of procedures are not necessary, but focus should be placed on procedural steps where personnel are vulnerable to exposure to potentially hazardous recombinant materials.

We will use lipid transient transfection methods to introduce multiple DNA plasmids containing the different viral genes necessary for propagating virus along with the shRNA/gRNA/gene of interest into HEK 293t cells. The viral genomes are broken into separate plasmids and several replication genes have been removed to ensure viral particles are replication incompetent. We will harvest and concentrate virus and other viral agents 48-72 hours post-transfection using syringes, filters, and ultracentrifugation methods. To titer the virus we will infect the appropriate host cell (e.g. HEK 293t cells for lentivirus) with a dilution of virus and then use flow cytometry to calculate the titer values. We will not be doing actual gene insertions/creation of gene constructs for other labs, or any other rDNA procedures (i.e. siRNA or other gene targeting, site-directed mutagenesis, etc.).

Please note that the viral vectors listed in the Vectors table are produced by the Core facility for researchers. The genes listed in the "Genes" table are those potentially packaged, and the hosts listed in the "Hosts" table include the hosts that the vectors can infect.





Vectors



Vector Name

Vector Type

Risk Group

Replication Incompetent

Amphotropic

Used in Animals

Adeno-associated Virus (AAV)

Viral

1

Yes

Yes

Yes

Avian Retrovirus

Viral

1

Yes

No

No

Lentivirus

Viral

2

Yes

Yes

No

Moloney Murine Leukemia Virus (MMLV)

Viral

2

Yes

Yes

No

various

Plasmid

1

No

No

No



Vector Name:

Adeno-associated Virus (AAV)

Risk Group:

1

Vector Type:

Viral

Replication incompetent:

Yes

Causes human disease:

No

Ecotropic:

No

Amphotropic:

Yes

Used in Animals:

Yes

Please indicate which generation of this viral vector you will use.

We will provide serotypes AAV1-6, and primarily package serotype 2 (the most commonly used AAV).

If you derive a new vector from wild-type virus, or similarly use an early generation vector (for example, first generation), please describe the method by which you will verify replication incompetence. Specify the frequency of testing.

N/A

Provide a detailed description of this vector and safety features of its design (such as which generation of viral vector, gene deletions, etc.):

AAV are infectious human viruses with no known disease association. Replication of AAV has an extremely low efficiency without the presence of helper virus (such as adenovirus). Similar to retrovirus such as HIV, packaging recombinant virus entails separating the replication and capsid genes and provided them transiently in trans (in a pRep/Cap plasmid), where only the 2 inverted terminal repeats of AAV genome are left and packaged into virion; hence, the likelihood for a recombinant AAV to convert to a replicative competency is highly improbably and some consider theoretically impossible. Recombinant AAV constructs in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus is in most cases be handled at BSL1.



Vector Name:

Avian Retrovirus

Risk Group:

1

Vector Type:

Viral

Replication incompetent:

Yes

Causes human disease:

No

Ecotropic:

Yes

Amphotropic:

No

Used in Animals:

No

Please indicate which generation of this viral vector you will use.




If you derive a new vector from wild-type virus, or similarly use an early generation vector (for example, first generation), please describe the method by which you will verify replication incompetence. Specify the frequency of testing.




Provide a detailed description of this vector and safety features of its design (such as which generation of viral vector, gene deletions, etc.):

Retroviral infection requires the specific interaction between the envelope glycoprotein on the surface of the virus and the cognate receptor on the surface of the cell. The ASLV family of viruses has five primary envelope types: A, B, C, D, E. These recognize three distinct cellular receptors: A, C, and B/D/E. In order for the virus to be propagated, your cells must have the appropriate receptor and cannot be sequentially infected with two ASLVs expressing the same envelope. Mammalian cells do not have functional receptors for the standard ASLV envelopes on their surface.



Vector Name:

Lentivirus

Risk Group:

2

Vector Type:

Viral

Replication incompetent:

Yes

Causes human disease:

Yes

Ecotropic:

No

Amphotropic:

Yes

Used in Animals:

No

Please indicate which generation of this viral vector you will use.

3rd generation

If you derive a new vector from wild-type virus, or similarly use an early generation vector (for example, first generation), please describe the method by which you will verify replication incompetence. Specify the frequency of testing.

N/A

Provide an explanation of the hazards to humans and describe the possible modes of transmission:

Lentivirus has potential to cause human disease if there were an exposure. We have placed a page describing safety considerations at the viracore website (http://viracore.ucsf.edu/content/biosafety).

Provide a detailed description of this vector and safety features of its design (such as which generation of viral vector, gene deletions, etc.):

Only "third generation" lentiviral vectors, such as the pSico or LentiLox vectors available from the UCSF Sandler Lentiviral RNAi Core. These systems separate vector and packaging functions onto three or four or more plasmids and they include additional safety features (e.g., they do not encode Tat, which is essential for replication of wild-type HIV-1).
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